Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells

ABSTRACT

The present disclosure relates to compositions and methods comprising cell surface markers for hES-derived cells, in particular, endoderm lineage cells including pancreatic endoderm-type cells, derived from hES cells.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/700,608, filed on Apr. 30, 2015, which is a continuation of U.S.patent application Ser. No. 13/724,556, filed on Dec. 21, 2012, issuedas U.S. Pat. No. 9,045,736, which is a continuation of U.S. patentapplication Ser. No. 12/107,020, filed on Apr. 21, 2008, issued as U.S.Pat. No. 8,338,170. The prior applications are incorporated by referencein their entirety.

REFERENCE TO SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled9511-96314-04_Sequence_Listing, created Oct. 28, 2016, which is 10.2 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates to a compositioncomprising a human embryonic stem cell-derived pancreatic endodermpopulation containing at least one cell type bearing at least onecellular target or marker and a method for purifying the same.

BACKGROUND

Human embryonic stem cells (hESCs) have the potential to producedifferentiated cell types comprising all human somatic tissues andorgans. Cell therapy treatment of insulin dependent diabetes isfacilitated by the production of unlimited numbers of pancreatic cellsthat can and will be able to function similarly to human islets.Accordingly, there is need for producing these pancreatic type cellsderived from hES cells, as well as reliable methods for purifying suchcells.

SUMMARY OF THE INVENTION

Embodiments described herein relate to methods for purifying varioushES-derived cell populations.

Additional embodiments of the invention are described in the followingnumbered paragraphs:

1. A method of purifying a gut endoderm cell comprising: a) exposing apopulation of cells derived from pluripotent stem cells comprising a gutendoderm cell to a ligand which binds to a cell surface marker expressedon the gut endoderm cell, wherein said cell surface marker is selectedfrom the group consisting of CD49e, CD99, CD165, and CD334; and b)separating the gut endoderm cell from cells derived from pluripotentstem cells which do not bind to the ligand, thereby purifying said gutendoderm cell.

2. The method of paragraph 1, wherein the ligand is an antibody orbinding fragment thereof.

3. The method of paragraph 2, wherein said antibody is a monoclonalantibody.

4. The method of paragraph 2, wherein said antibody is a polyclonalantibody.

5. The method of paragraph 1, wherein the gut endoderm cell expresses atleast forkhead box A2 (FOXA2).

6. A method of purifying a gut endoderm cell comprising: a) exposing apopulation of cells derived from pluripotent stem cells comprising a gutendoderm cell to a ligand which binds to a cell surface marker notexpressed on said gut endoderm cell, wherein the cell surface marker isselected from the group consisting of CD30, CD49a and CD55; and b)separating the gut endoderm cell from cells derived from pluripotentstem cells which bind to the ligand, thereby purifying said gut endodermcell.

7. The method of paragraph 6, wherein the gut endoderm cell expresses atleast forkhead box A2 (FOXA2).

8. The method of paragraph 6, wherein the ligand is an antibody orbinding fragment thereof.

9. The method of paragraph 8, wherein said antibody is a monoclonalantibody.

10. The method of paragraph 8, wherein said antibody is a polyclonalantibody.

11. A method of purifying a pancreatic endoderm cell comprising: a)exposing a population of cells derived from pluripotent stem cellscomprising a pancreatic endoderm cell to a ligand which binds to a cellsurface marker on the pancreatic endoderm cell, wherein the cell surfacemarker is selected from the group consisting of CD57 and CD142, andwherein the pancreatic endoderm cell expresses pancreatic and duodenalhomeobox gene 1 (PDX1); and b) separating the pancreatic endoderm cellfrom cells derived from pluripotent stem cells which do not bind to theligand, thereby purifying said pancreatic endoderm cell.

12 The method of paragraph 11, wherein the ligand is an antibody orbinding fragment thereof.

13. The method of paragraph 12, wherein said antibody is a monoclonalantibody.

14. The method of paragraph 12, wherein said antibody is a polyclonalantibody.

15. The method of paragraph 11, wherein the pancreatic endoderm cellexpresses NKX6.1.

16. A method of purifying a pancreatic endocrine cell comprising: a)exposing a population of cells derived from pluripotent stem cellscomprising a pancreatic endocrine cell to a ligand which binds to a cellsurface marker expressed on the pancreatic endocrine cell, wherein thecell surface marker is selected from the group consisting of CD57,CD200, and CD318; and b) separating the pancreatic endocrine cell fromcells derived from pluripotent stem cells which do not bind to theligand, thereby purifying said pancreatic endocrine cell.

17. The method of paragraph 16, wherein the pancreatic endocrine cellexpresses at least chromogranin A (CHGA).

18. The method of paragraph 16, wherein the ligand is an antibody orbinding fragment thereof.

19. The method of paragraph 18, wherein said antibody is a monoclonalantibody.

20. The method of paragraph 18, wherein said antibody is a polyclonalantibody.

21. A method of purifying a pancreatic endocrine cell comprising: a)exposing a population of cells derived from pluripotent stem cellscomprising a pancreatic endocrine cell to a ligand which binds to a cellsurface marker not expressed on said pancreatic endocrine cell, whereinthe cell surface marker is selected from the group consisting of CD142and CD340; and b) separating the pancreatic endocrine cell from cellsderived from pluripotent stem cells which bind to the ligand, therebypurifying said pancreatic endocrine cell.

22. The method of paragraph 21 wherein the pancreatic endocrine cellexpresses at least chromogranin A (CHGA).

23. The method of paragraph 21, wherein the ligand is an antibody orbinding fragment thereof.

24. The method of paragraph 23, wherein said antibody is a monoclonalantibody.

25. The method of paragraph 23, wherein said antibody is a polyclonalantibody.

26. A method of purifying a pancreatic endoderm cell comprising: a)exposing a population of cells derived from pluripotent stem cellscomprising a pancreatic endoderm cell to a ligand which binds to a cellsurface marker not expressed on said pancreatic endoderm cell, whereinsaid cell surface marker is selected from the group consisting of CD55and CD98, and wherein the pancreatic endoderm cell expresses pancreaticand duodenal homeobox gene 1 (PDX1); and b) separating the pancreaticendoderm cell from cells derived from pluripotent stem cells which bindto the ligand, thereby purifying said pancreatic endoderm cell.

27. The method of paragraph 26, wherein the ligand is an antibody orbinding fragment thereof.

28. The method of paragraph 27, wherein said antibody is a monoclonalantibody.

29. The method of paragraph 27, wherein said antibody is a polyclonalantibody.

30. The method of paragraph 27, wherein the pancreatic endoderm cellexpresses NKX6.1.

31. A purified cell population comprising human gut endoderm cells.

32. The purified population of paragraph 31, wherein said human gutendoderm cells comprise at least 50% of the human cells in said cellpopulation.

33. The purified population of paragraph 31, wherein said human gutendoderm cells comprise at least 75% of the human cells in said cellpopulation.

34. The purified population of paragraph 31, wherein said human gutendoderm cells express at least forkhead box A2 (FOXA2).

35. A purified cell population comprising human pancreatic endodermcells.

36. The purified population of paragraph 35, wherein said humanpancreatic endoderm cells comprise at least 50% of the human cells insaid cell population.

37. The purified population of paragraph 35, wherein said humanpancreatic endoderm cells comprise at least 75% of the human cells insaid cell population.

38. The purified population of paragraph 35, wherein said humanpancreatic endoderm cells express pancreatic and duodenal homeobox gene1 (PDX1) and NKX6.1.

39. A purified cell population comprising human pancreatic endocrinecells.

40. The purified population of paragraph 39, wherein said humanpancreatic endocrine cells comprise at least 50% of the human cells insaid cell population.

41. The purified population of paragraph 39, wherein said humanpancreatic endocrine cells comprise at least 75% of the human cells insaid cell population

42. The purified population of paragraph 39, wherein said humanpancreatic endocrine cells express at least chromogranin A (CHGA).

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.

Processes and compositions related to but distinct from the presentinvention can be found in U.S. Provisional Patent Application No.60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2003; U.S.Provisional Patent Application No. 60/566,293, entitled PDX1 EXPRESSINGENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent Application No.60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATIONOF DEFINITIVE ENDODERM, filed Jul. 9, 2004; U.S. Provisional PatentApplication No. 60/587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FORTHE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14, 2004; U.S. patentapplication Ser. No. 11/021,618, entitled DEFINITIVE ENDODERM, filedDec. 23, 2004 and U.S. patent application Ser. No. 11/115,868, entitledPDX1 EXPRESSING ENDODERM, filed Apr. 26, 2005; U.S. patent applicationSer. No. 11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FORDIFFERENTIATING DEFINITIVE ENDODERM, filed Jun. 23, 2005; U.S.Provisional Patent Application No. 60/730,917, entitled PDX1-EXPRESSINGDORSAL AND VENTRAL FOREGUT ENDODERM, filed Oct. 27, 2005; U.S.Provisional Patent Application No. 60/736,598, entitled MARKERS OFDEFINITIVE ENDODERM, filed Nov. 14, 2005; U.S. Provisional PatentApplication No. 60/778,649, entitled INSULIN-PRODUCING CELLS AND METHODOF PRODUCTION, filed Mar. 2, 2006; U.S. Provisional Patent ApplicationNo. 60/833,633, entitled INSULIN-PRODUCING CELLS AND METHOD OFPRODUCTION, filed Jul. 26, 2006; U.S. Provisional Patent Application No.60/852,878, entitled ENRICHMENT OF ENDOCRINE PRECURSOR CELLS, IMMATUREPANCREATIC ISLET CELLS AND MATURE PANCREATIC ISLET CELLS USING NCAM,filed Oct. 18, 2006; U.S. patent application Ser. No. 11/588,693,entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed Oct.27, 2006; U.S. patent application Ser. No. 11/681,687, entitledENDOCRINE PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS ANDMETHODS OF PRODUCTION, filed Mar. 2, 2007; U.S. patent application Ser.No. 11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES, filedJul. 5, 2007; U.S. Patent Application No. 60/972,174, entitled METHODSOF TREATMENT FOR DIABETES, filed Sep. 13, 2007; U.S. patent applicationSer. No. 11/860,494, entitled METHODS FOR INCREASING DEFINITIVE ENDODERMPRODUCTION, filed Sep. 24, 2007; and U.S. Patent Application No.60/977,349, entitled CELL SURFACE MARKERS OF HUMAN EMBRYONIC STEM CELLSAND CANCER STEM CELLS, filed Oct. 3, 2007, the disclosures of which areincorporated herein by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representing five-stage differentiation protocolfrom ES cells (ES) though mesendoderm (ME) to definitive endoderm (DE or“PDX1-negative definitive endoderm”), primitive gut tube (PG or“PDX1-negative foregut endoderm”), posterior foregut (PF or“PDX1-positive foregut endoderm), pancreatic endoderm (PE), and finallyendocrine (EN) cells. The diagram also describes “signature” markersexpressed in each cell type as well as selected cell surface markersthat can be used to enrich or purify the indicated cell type.

FIG. 2 is a pie chart describing the percentage composition of CHGA+endocrine cells, CHGA−/NKX6.1+/PDX1+ pancreatic endoderm cells, PDX1+endoderm cells that are CHGA−/NKX6.1− and other cells types in a stage4/5 cell culture.

DETAILED DESCRIPTION

Described herein are methods and compositions for identifying andcharacterizing various hES-derived cell types using various antibodieswhich cross-react to cell surface markers, receptors, membrane proteinsand/or epitopes.

Also described herein is the use of hES-derived cell types, which weremade from a progression of steps for converting undifferentiated hESCsto hESC-derived cells, for example, pancreatic endoderm or epithelialcells, endocrine precursor or progenitor cells, and/or hormone secretingendocrine cells. This progression of steps directs the sequentialdifferentiation of hESCs through intermediates that are currentlyrecognized to occur during pancreatic development in vivo. Generalmethods for production of hESC-derived cells are described in relatedU.S. applications as indicated above, and d'Amour et al. 2005 NatBiotechnol. 23:1534-41, D'Amour et al. 2006 Nat Biotechnol.24(11):1392-401 the disclosures of which are incorporated herein byreference in their entireties.

DEFINITIONS

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,recombinant DNA and immunology, which are within the capabilities of aperson of ordinary skill in the art. Such techniques are explained inthe literature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.(1995 and periodic supplements; Current Protocols in Molecular Biology,ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; J. M. Polak and James O′D. McGee, 1990,In Situ Hybridization: Principles and Practice; Oxford University Press;M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A PracticalApproach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methodsof Enzymology: DNA Structure Part A: Synthesis and Physical Analysis ofDNA Methods in Enzymology, Academic Press; and E. M. Shevach and W.Strober, 1992 and periodic supplements, Current Protocols in Immunology,John Wiley & Sons, New York, N.Y., Current Protocols in Cytometry, JohnWiley & Sons, New York, N.Y. Each of these general texts is hereinincorporated by reference in its entirety.

It will be appreciated that the numerical ranges expressed hereininclude the endpoints set forth and describe all integers between theendpoints of the stated numerical range.

In some embodiments, hESCs can be derived from a “pre-implantationembryo.” As used herein, “pre-implantation embryo” refers to an embryobetween the stages of fertilization and implantation. Thus, apre-implantation embryo typically has not progressed beyond theblastocyst stage. Implantation usually takes place 7-8 days afterfertilization. However, implantation can take place about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14 or greater than about 14 days afterfertilization.

As used herein, “multipotent” or “multipotent cell” refers to a celltype that can give rise to a limited number of other particular celltypes. Multipotent cells are committed to one or more embryonic cellfates, and thus, in contrast to pluripotent cells, cannot give rise toeach of the three embryonic cell lineages as well as extraembryoniccells.

In some embodiments, “pluripotent cells” are used as the startingmaterial for pancreatic islet hormone-expressing cell differentiation.By “pluripotent” is meant that the cell can give rise to each of thethree embryonic cell lineages as well as extraembryonic cells.Pluripotent cells, however, may not be capable of producing an entireorganism.

In certain embodiments, the pluripotent cells used as starting materialare stem cells, including human embryonic stem cells. As used herein,“embryonic” refers to a range of developmental stages of an organismbeginning with a single zygote and ending with a multicellular structurethat no longer comprises pluripotent or totipotent cells other thandeveloped gametic cells. In addition to embryos derived by gametefusion, the term “embryonic” refers to embryos derived by somatic cellnuclear transfer.

As used herein, “human embryonic stem cells” or “hES cells” or “hESCs”or “stem cells” or “pluripotent stem cells” are cells obtained from ananimal (e.g., a primate, such as a human) embryo. These terms andphrases are equivalent to the phrase, “differentiable cell”. A“differentiable cell” is used to describe a cell or population of cellsthat can differentiate into at least partially mature cells, or that canparticipate in the differentiation of cells, e.g., fuse with othercells, that can differentiate into at least partially mature cells.

As used herein, the phrase, “differentiable cell” or “differentiatedcell” or “hES-derived cell” can refer to pluripotent, multipotent,oligopotent or even unipotent cells, as defined in detail below. Incertain embodiments, the differentiable cells are pluripotentdifferentiable cells. In more specific embodiments, the pluripotentdifferentiable cells are selected from the group consisting of embryonicstem cells, ICM/epiblast cells, primitive ectoderm cells, primordialgerm cells, and teratocarcinoma cells. In one particular embodiment, thedifferentiable cells are mammalian embryonic stem cells. In a moreparticular embodiment, the differentiable cells are human embryonic stemcells. Of course, certain embodiments also contemplate differentiablecells from any source within an animal, provided the cells aredifferentiable as defined herein. For example, differentiable cells canbe harvested from embryos, or any primordial germ layer therein, fromplacental or chorion tissue, or from more mature tissue such as adultstem cells including, but not limited to adipose, bone marrow, nervoustissue, mammary tissue, liver tissue, pancreas, epithelial, respiratory,gonadal and muscle tissue. In specific embodiments, the differentiablecells are embryonic stem cells. In other specific embodiments, thedifferentiable cells are adult stem cells. In still other specificembodiments, the stem cells are placental- or chorionic-derived stemcells.

As used herein, “partially mature cells” are cells that exhibit at leastone characteristic of the phenotype, such as morphology or proteinexpression, of a mature cell from the same organ or tissue. Someembodiments contemplate using differentiable cells from any animalcapable of generating differentiable cells, e.g., pancreatic type cellssuch as beta cells. The animals from which the differentiable cells areharvested can be vertebrate or invertebrate, mammalian or non-mammalian,human or non-human. Examples of animal sources include, but are notlimited to, primates, rodents, canines, felines, equines, bovines andporcines.

As used herein, the term “differentiate” refers to the production of acell type that is more differentiated than the cell type from which itis derived. The term therefore encompasses cell types that are partiallyand terminally differentiated. Similarly, “produced from hESCs,”“derived from hESCs,” “differentiated from hESCs,” “h-ES derived cell”and equivalent expressions refer to the production of a differentiatedcell type from hESCs in vitro and in vivo.

As used herein, “definitive endoderm” or “DE” refers to a multipotentcell that can differentiate into cells of the gut tube or organs derivedfrom the gut tube. In accordance with certain embodiments, thedefinitive endoderm cells and cells derived therefrom are mammaliancells, and in a preferred embodiment, the definitive endoderm cells arehuman cells. In some embodiments, definitive endoderm cells express orfail to significantly express certain markers. In some embodiments, oneor more markers selected from SOX17, CXCR4, MIXL1, GATA4, FOXA2, GSC,FGF17, VWF, CALCR, FOXQ1, CMKOR1, CER and CRIP1 are expressed indefinitive endoderm cells. In other embodiments, one or more markersselected from OCT4, HNF4A, alpha-fetoprotein (AFP), Thrombomodulin (TM),SPARC and SOX7 are not significantly expressed in definitive endodermcells. Definitive endoderm does not express PDX-1. See “DE” in FIG. 1.

Still other embodiments relate to cell cultures termed “PDX1-negative(PDX1−) foregut endoderm cells”, “primitive gut (PG) tube endodermcell”, “gut endoderm”, “foregut endoderm”, or equivalents thereof. See“PG” in FIG. 1. PDX1-negative foregut endoderm cells are multipotentcells of the definitive endoderm lineage and have high SOX17, HNF4alpha(HNF4A), HNF1beta (HNF1B), FOXA1, FOXA2 expression; and low orsignificantly no PDX1, AFP, SOX7, SOX1, ZIC1 and NFM expression.

Other embodiments relate to cell cultures termed “PDX1-positive,dorsally-biased, foregut endoderm cells”, “PDX1-positive foregut (PF)endoderm cells”, “PDX1 positive endoderm”, “pancreatic endoderm (PE)” orequivalents thereof. In some embodiments, the PDX1-positive foregutendoderm cells express one or more markers including PDX1, HNF6, PROX1and SOX9. See “PF” in FIG. 1. In other embodiments, PDX1-positiveforegut endoderm cells are further differentiated to other, at least,pancreatic-lineage cells, for example, PDX1-positive (PDX1+) endodermwhich does not express NKX6.1 or CHGA (PDX1+/NKX6.1−/CHGA−) andPDX1-positive cells which do express NKX6.1 but do not express CHGA(PDX1+/NKX6.1+/CHGA−). See “PE” and “PDX1” cells/circles in FIG. 1.Hence, in one embodiment, a PDX1-positive foregut endoderm cell precedesa PDX1+/NKX6.1− and a PDX1+/NKX6.1+ type cell. In other aspects,PDX1+/NKX6.1− endoderm cells transition to PDX1+/NKX6.1+ endoderm cells.Both PDX1+/NKX6.1− and PDX1+/NKX6.1+ endoderm cells do not expresshormone genes (e.g., insulin, glucagon, and the like); and are said tobe CHGA− negative (CHGA−). However, at least a small percentage of thesePDX1-positive foregut endoderm cells (PF) can further differentiate tonon-pancreatic-lineage type cells, e.g., posterior stomach and duodenum.

Both PDX1-negative foregut and PDX-1 positive foregut endoderm cellpopulations are further described in detail in related U.S. applicationSer. No. 11/588,693 entitled PDX1 EXPRESSING DORSAL AND VENTRAL FOREGUTENDODERM, filed Oct. 27, 2006, which is herein incorporated by referencein its entirety. For example, Tables 3 and 4 of U.S. application Ser.No. 11/588,693 describes various markers in PDX1-positive foregutendoderm cells.

As described above, PDX1-positive foregut endoderm cells can furtherdifferentiate into other, at least, pancreatic-lineage type cells, e.g.,PDX1+/NKX6.1+/CHGA− and/or PDX1+/NKX6.1−/CHGA− cells. In a preferredembodiment, PDX1+/NKX6.1+/CHGA− cells further differentiate to apancreatic-lineage cell expressing PDX1 and neurogenin 3 (NGN3); and notstomach and/or duodenum NGN3-positive cells. The pancreatic-lineageNGN3-positive (NGN3+) cells can also be referred to as “endocrineprogenitor (EP)”, “endocrine precursor” cells. See “EP” in FIG. 1.Endocrine progenitors are multipotent cells of the definitive endodermlineage and although they cannot differentiate into as many differentcell, tissue and/or organ types as compared to less specificallydifferentiated definitive endoderm lineage or PDX1-positive foregutendoderm cells, they can differentiate to an endocrine or a hormonesecreting cell.

Still other embodiments relate to cell cultures or populationscontaining “endocrine cells”, “hormone secreting cells” or equivalentsthereof which refer to a cell expressing one or more pancreatic hormonesand is capable of has at least some of the functions of a humanpancreatic islet cell. Pancreatic islet hormone-expressing cells can bemature or immature. As used herein, the term “mature” refers to adifferentiated hES-derived cell, for example, an endocrine cell which iscapable of secreting a hormone and is also responsive to various agentsor insults which stimulates or effects the secretion of such hormone, invitro or in vivo. For example, a mature endocrine cell as describedherein is a cell which is, for example, glucose responsive. That is, invitro or in vivo, a mature endocrine cell, e.g., a pancreatic beta cell,which is sensitive to glucose and can secrete and release insulin in aphysiologically appropriate manner. As used herein, the term “immature”refers to a differentiated hES-derived cell, which in some embodiments,is not fully capable of secreting hormones, or, in other embodiments,not fully capable of secreting hormones due to various stimuli in vitro.Additionally, markers of mature and/or immature pancreatic islethormone-expressing cells or endocrine cells include, but are not limitedto, PDX1, ghrelin (GHRL), islet amyloid polypeptide (IAPP), insulin(INS), glucagon (GCG), NKX6 transcription factor related, locus 1(NKX6.1), somatostatin (SOM; SST), pancreatic polypeptide (PP);synaptophysin (SYP), glucokinase, (GCK), Chromogranin A (CHGA) and/orconnecting peptide (C-peptide). These and other markers of endocrinecells are described in related U.S. patent application Ser. No.11/773,944 entitled METHOD FOR PRODUCING PANCREATIC HORMONES, filed Jul.5, 2007 which is herein incorporated by reference in its entirety. Themature or immature pancreatic islet hormone-expressing cells produced bythe processes described herein express one or more of the above-listedmarkers, thereby producing the corresponding gene products. However, itwill be appreciated that mature pancreatic islet hormone-expressingcells need not express all of the above-described markers. For example,pancreatic islet hormone-expressing cells differentiated from hESCs donot co-express INS and GHRL. This pattern of gene expression isconsistent with the expression of these genes in human fetal pancreas.

In certain embodiments, the terms “enriched”, “isolated”, “separated”,“sorted”, “purified” or equivalents thereof refer to a cell culture or acell population or cell sample that contains at least about 30%, 40%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the desiredcell lineage or a desired cell having a certain cell phenotype, e.g.,expressing a certain cell marker or not expressing a certain cell markergene characteristic of that cell phenotype.

Many stem cell media culture or growth environments are embodied herein,including, but not limited to, defined media, conditioned media,feeder-free media, serum-free media and the like. As used herein, theterm “growth environment” or “milieu” or equivalents thereof is anenvironment in which stem cells (e. g., primate embryonic stem cells)will proliferate in vitro. Features of the environment include themedium in which the cells are cultured, and a supporting structure (suchas a substrate on a solid surface) if present.

As used herein, the term “enzymatically dissociated” or “enzymaticallytreated” or equivalents thereof refer to dissociating clusters orembryoid bodies of hES cells or hES-derived cells into single cellpopulations. In certain embodimentscells are dissociated to single cellpreparations, e.g., cell separation by flow cytometry (FACS) and/ordispensing of live hES cells into multi-well plates for use in highthroughput screening. Cowan et al., describe enzymatic dissociation ofhES cells using trypsin but they are propagated as small clusters ratherthan single cells. See Cowan et al., New Engl. J. Med. 350:1353-1356,2004; and Hasegawa et al., Stem Cells 24:2649-2660, 2006. In oneembodiment, hES cells or hES-derived cells are enzymatically treated ordissociated using Accutase. Dissociated cells can be grown to a highdensity as monolayers, and still retain their pluripotency, in the caseof undifferentiated hES cells. Use of Accutase is also described inPCT/US2007/62755, entitled COMPOSITIONS AND METHODS USEFUL FOR CULTURINGDIFFERENTIABLE CELLS, filed Feb. 23, 2007, which is herein incorporatedin its entirety by reference.

As used herein, the term “defined medium” or “defined media” orequivalents thereof refers to a biochemically defined formulationcomprised solely of the biochemically-defined constituents. In someembodiments, a defined medium includes solely constituents having knownchemical compositions. In other embodiments, a defined medium includesconstituents that are derived from known sources. For example, a definedmedium can also include factors and other compositions secreted fromknown tissues or cells; however, the defined medium will not include theconditioned medium from a culture of such cells. Thus, a “definedmedium” can, if indicated, include particular compounds added to formthe culture medium. Defined media compositions are known in the art, forexample, PCT/US2007/062755 filed Jun. 13, 2007 and marketed asStemPro®hESC SFM by Invitrogen, Carlsbad, Calif., which is hereinincorporated in its entirety.

As used herein, the phrase “conditioned medium” refers to a medium thatis altered as compared to a base medium. For example, the conditioningof a medium can cause molecules, such as nutrients and/or growthfactors, to be added to or depleted from the original levels found inthe base medium. In some embodiments, a medium is conditioned byallowing cells of certain types to be grown or maintained in the mediumunder certain conditions for a certain period of time. For example, amedium can be conditioned by allowing hESCs to be expanded,differentiated or maintained in a medium of defined composition at adefined temperature for a defined number of hours. As will beappreciated by those of skill in the art, numerous combinations ofcells, media types, durations and environmental conditions can be usedto produce nearly an infinite array of conditioned media.

As used herein, the term “feeder cells” or “feeder cell layers” arecells which grow in vitro and are co-cultured with a target cell, e.g.,co-cultured with stem cells. As used herein, the term “essentially freeof a feeder cell” or “feeder-free” and equivalents thereof, refer totissue culture conditions that do not contain intentionally added feedercells. Also, a cell culture is “essentially feeder-free” when it doesnot contain exogenously added conditioned medium taken from a culture offeeder cells or exogenously added feeder cells. In some embodiments, “noexogenously added feeder cells” means that cells capable of developing afeeder cell layer have not been purposely introduced for that reason. Ofcourse, if the cells to be cultured are derived from a seed culture thatcontained feeder cells, the incidental co-isolation and subsequentintroduction into another culture of some small proportion of thosefeeder cells along with the desired cells (e. g., undifferentiatedprimate stem cells) should not be deemed as an intentional introductionof feeder cells. In such an instance, the culture contains a de minimusnumber of feeder cells. By “de minimus”, it is meant the number oramount of feeder cells that is carried from a first culture to a secondculture where the cells of the first culture have been cultured onfeeder cells. Similarly, feeder cells or feeder-like cells that developfrom stem cells seeded into the culture shall not be deemed to have beenpurposely introduced into the culture.

As used herein, the term “basal medium” refers to a solution of aminoacids, vitamins, salts, and nutrients that is effective to support thegrowth of cells in culture, although normally these compounds will notsupport cell growth unless supplemented with additional compounds. Thenutrients include a carbon source (e.g., a sugar such as glucose) thatcan be metabolized by the cells, as well as other compounds necessaryfor the cells' survival. These are compounds that the cells themselvescannot synthesize, due to the absence of one or more of the gene(s) thatencode the protein(s) necessary to synthesize the compound (e.g.,essential amino acids) or, with respect to compounds which the cells cansynthesize, because of their particular developmental state the gene(s)encoding the necessary biosynthetic proteins are not being expressed assufficient levels. A number of base media are known in the art ofmammalian cell culture, such as Dulbecco's Modified Eagle Media (DMEM),Knockout-DMEM (KO-DMEM), and DMEM/F12, although any base medium thatsupports the growth of primate embryonic stem cells in a substantiallyundifferentiated state can be employed. A “basal medium” as describedherein also refers to the basal medium described in PCT/US2007/062755,filed Jun. 13, 2007, which is herein incorporated in its entirety.

In some embodiments the basal medium contains “exogenous insulin orinsulin substitutes”, which refers to insulin or insulin substitutesthat is/are not intentionally added to the compositions or methods. Inother embodiments, the basal medium does not contain “exogenous insulinor insulin substitutes.” Thus, in certain embodiments, the methods andcompositions are free of insulin or insulin substitutes that areintentionally supplied. In other embodiments, the compositions ormethods may, however, not necessarily be free of endogenous insulin. Asused herein, “endogenous insulin” indicates that the cultured cellsproduce insulin when cultured according to the embodiments describedherein. In some embodiments, endogenous insulin is used to detectresidual impurities from the primary cell culture or impurities from thestarting materials. In some embodiments, the compositions contain lessthan 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 μg/ml, or 0.7 to 7 ng/ml of insulinor substantially no amounts of insulin.

Moreover, it has been demonstrated that high insulin concentration,e.g., concentrations as little as 0.2 μg/ml, is detrimental for theproduction of definitive endoderm cells from hES cells. See McLean etal. Stem Cells 25:29-38 2007. Contacting the cells with insulin duringthe TGFbeta stimulated differentiation process keeps the cells in thepluripotent undifferentiated state (Step 1 of d'Amour et al. 2006,supra). Hence, presence of high insulin can promote production of otherhES-derived cells types other than definitive endoderm, which productionis necessary for the further production of other endodermally-derived,hES-derived, cell types described herein. Production of hES-derivedcells in a culture medium containing Knockout serum (GIBCO BRL) orStemPro34 (GIBCO BRL) supplement contains about 8-16 μg/ml and 15 μg/mlof insulin respectively. See U.S. Publication 2006/0003446 to Keller etal. In contrast, hES-derived cells produced herein, in particularproduction of definitive endoderm cells, lack substantial amounts ofinsulin, e.g., the ranges described herein and in the relatedapplications indicated above, describe production of definitive endodermin a culture having about 0.7 ng to 7 ng/ml of insulin.

To be clear, the term “insulin” refers to the protein, or variant orfragment thereof that binds to the insulin receptor in normalphysiological concentrations and can induce signaling through theinsulin receptor. The term “insulin” encompasses a protein having thepolypeptide sequence of native human insulin, or of other mammalianinsulin, or of any homologs or variants to these sequences.Additionally, the term insulin encompasses polypeptide fragments thatare capable of binding to the insulin receptor to induce signalingthrough the insulin receptor. The term “insulin substitute” refers toany zinc containing compound that can be used in place of insulin togive substantially similar results as insulin. Examples of insulinsubstitutes include, but are not limited to zinc chloride, zinc nitrate,zinc bromide, and zinc sulfate.

Additionally, insulin-like growth factors are not insulin substitutes orhomologs of insulin, as contemplated in the embodiments presentedherein. However, it will be appreciated that IGF-1 will inhibitdefinitive endoderm formation at even lower concentrations than insulin.See McLean et al. Stem Cells 25:29-38 2007. Accordingly, in anotherspecific embodiment, the compositions and methods can comprise the useof at least one insulin-like growth factor (IGF) or a variant or afunctional fragment thereof. Still, in other embodiments, thecompositions and methods described herein are free of any exogenousinsulin-like growth factors (IGFs). In specific embodiments, thecompositions and methods described herein contain less than about 200,150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml ofIGF-1.

The culture conditions described herein are “isotonic”, which termrefers to a solution having essentially the same tonicity (i.e.,effective osmotic pressure equivalent) as another solution with which itis compared. In the context of cell culture, an “isotonic” medium is onein which cells can be cultured without an appreciable net flow of wateracross the cell membranes.

When used in connection with cell cultures and/or cell populations, theterm “portion” means any non-zero amount of the cell culture or cellpopulation, which ranges from a single cell to the entirety of the cellculture or cells population. In preferred embodiments, the term“portion” means at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, at least 13%, atleast 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94% or at least 95% of the cell culture or cell population.

With respect to cells in cell cultures or in cell populations, the term“substantially free of” or “essentially free of” means that thespecified cell type of which the cell culture or cell population isfree, is present in an amount of less than about 10%, less than about9%, less than about 8%, less than about 7%, less than about 6%, lessthan about 5%, less than about 4%, less than about 3%, less than about2% or less than about 1% of the total number of cells present in thecell culture or cell population.

As used herein, “marker”, “epitope”, “target”, “receptor” or equivalentsthereof can refer to any molecule that can be observed or detected. Forexample, a marker can include, but is not limited to, a nucleic acid,such as a transcript of a specific gene, a polypeptide product of agene, such as a membrane protein, a non-gene product polypeptide, aglycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein or asmall molecule (for example, molecules having a molecular weight of lessthan 10,000 amu). A “cell surface marker” is a marker present at thecell surface.

As used herein, “ligand” refers to a moiety or binding partner thatspecifically binds or cross-reacts to the marker or target or receptoror membrane protein on the cell or to the soluble analyte in a sample orsolution. The target on the cell, includes but is not limited to amarker. Examples of such ligands include, but are not limited to, anantibody that binds a cellular antigen, an antibody that binds a solubleantigen, an antigen that binds an antibody already bound to the cellularor soluble antigen; a lectin that binds to a soluble carbohydrate or toa carbohydrate moiety which is a part of a glycoprotein or glycolipid;or functional fragments of such antibodies and antigens that are capableof binding; a nucleic acid sequence sufficiently complementary to atarget nucleic acid sequence of the cellular target or soluble analyteto bind the target or analyte sequence, a nucleic acid sequencesufficiently complementary to a ligand nucleic acid sequence alreadybound to the cellular marker or target or soluble analyte, or a chemicalor proteinaceous compound, such as biotin or avidin. Ligands can besoluble or can be immobilized on the capture medium (i.e., syntheticallycovalently linked to a bead), as indicated by the assay format, e.g.,antibody affinity chromatography. As defined herein, ligands include,but are not limited to, various agents that detect and react with one ormore specific cellular markers or targets or soluble analytes. Examplesof ligands are those described herein which selectively bind to a targetand/or epitope including, but without limitation, CD30, CD49a, CD49e,CD55, CD99, CD165, CD334, CD57, CD98, CD142, CD200, CD318, CD340 or anyof the ligands and/or agents and/or antibodies which selectively bind tothose targets described in Table 1 (Example 1). Further, all suchligands are characterized by the desired ability to bind the specifiedmarker or target or analyte, whether it is soluble or bound to a cell.In one preferred embodiment, the ligand is a component thatpreferentially binds to all or a portion of a cell surface receptor.Thus, a ligand useful in this embodiment can be an antibody, or afragment thereof, capable of binding to a cell surface receptor on a hESor hES-derived cell.

As used herein, the terms “contacting” or “exposing” or equivalentsthereof refer to combining or mixing. For example, putative IgG, IgM,IgA, IgD, IgE or hybrids, derivatives or fragments of any of theaforementioned antibodies, can be contacted with a hES-derived cellpopulation, including a population containing endoderm lineage cellsdescribed in Stages/Steps 1-5 of D'Amour et al. 2005 & 2006, supra andas represented in FIG. 1 as well as U.S. patent application Ser. No.11/773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES, filedJul. 5, 2007, the disclosure of which is incorporated herein byreference in its entirety. In some embodiments, formation of a complexbetween the hES-derived cell and the IgG, IgM, IgA, IgD IgE or hybrids,derivatives or fragments of any of the aforementioned antibody moleculesrefers to the ability of the target, receptor or membrane protein toselectively bind to the immunoglobulin molecule, or binding portionthereof, in order to form a stable complex that can be measured (i.e.,detected) or quantified. Selective binding between a the target,receptor or membrane protein and an immunoglobulin molecule, or bindingfragment thereof, for example, is effected under conditions suitable toform a complex; such conditions (e.g., appropriate concentrations,buffers, temperatures, reaction times) as well as methods to optimizesuch conditions are known to those skilled in the art, and examples aredisclosed herein. Examples of complex formation conditions are alsodisclosed in, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989, the referenceSambrook et al., ibid., is incorporated by reference herein in itsentirety.

As used herein, the term “detecting complex formation” refers todetermining if any complex is formed, i.e., assaying for the presence(i.e., existence) of a complex. If complexes are formed, the amount ofcomplexes formed can, but need not be, determined. Complex formation, orselective binding, between the target, receptor and/or membrane proteinand any immunoglobulin molecule in the composition can be measured(i.e., detected, determined) using a variety of methods standard in theart (see, for example, Sambrook et al. supra), examples of which aredisclosed herein.

As used herein, the term, “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immuno reacts with) an antigen. Such antibodies or fragmentsinclude polyclonal antibodies from any native source, and native orrecombinant monoclonal antibodies of classes IgG, IgM, IgA, IgD, andIgE, hybrid derivatives, and fragments of antibodies including Fab, Fab′and F(ab′)2, humanized or human antibodies, recombinant or syntheticconstructs containing the complementarity determining regions of anantibody, an Fc antibody fragment thereof, a single chain Fv antibodyfragment, a synthetic antibody or chimeric antibody construct whichshares sufficient CDRs to retain functionally equivalent bindingcharacteristics of an antibody that binds a desired cell surfacereceptor, and a binding fragment produced by phage display. Certainclasses have subclasses as well, such as IgG1, IgG2, and others.Furthermore, in humans, the light chain can be a kappa chain or a lambdachain. Reference herein to antibodies includes a reference to all suchclasses, subclasses and types of human antibody species. Antibodies usedin the examples described herein were generally obtained by conventionalhybridoma methods and purified from ascites fluid by ammonium sulfate(45%) precipitation, centrifugation and affinity chromatography usingprotein A. The standard process of making monoclonal antibodies isdescribed in G. Kohler and C. Milstein, 1975 Nature, 256: 495-497. Ofcourse, the particular method of making and the type of monoclonalantibody is not limited to such techniques and it is envisioned that anytechnique for making such antibodies is within the practice of theembodiments described herein.

Where indicated, the ligands and/or agents and/or antibodies employed inthe embodiments described herein are associated with detectable labels.Hence, as used herein, the term “label” or “detectable label” refers to,for example, radioactive, fluorescent, luminescent, chemiluminescent,biological or enzymatic tags or labels of standard use in the art.Detectable labels for attachment to components useful in certainembodiments described herein can be easily selected from among numerouscompositions known and readily available to one skilled in the art ofdiagnostic assays. In some embodiments, the label can be a smallchemical molecule that is capable, acting alone, or in concert withother molecules or proteins, of providing a signal, that is detectableeither directly or indirectly. In preferred embodiments, the marker ortarget is associated with the various ligands or competing analytes usedin the assays. The reagents, ligands, competing analytes, or capturemedium described herein are not limited by the particular detectablelabel or label system employed. In some cases, the detectable label caninclude the refractive index of a cell surface or bead. A detectablelabel can be conjugated, or otherwise bound, to nucleic acids,polypeptides, such as antibodies, or small molecules. For example,oligonucleotides described herein can be labeled subsequent tosynthesis, by incorporating biotinylated dNTPs or rNTP, or some similarmeans (e.g., photo-cross-linking a protein derivative of biotin toRNAs), followed by addition of labeled streptavidin (e.g.,phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively,when fluorescently-labeled oligonucleotide probes are used, fluorescein,lissamine, phycoerythirin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3,Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others, can be attached tonucleic acids. Non-limiting examples of detectable labels that can beconjugated to polypeptides such as antibodies include but are notlimited to radioactive labels, such as ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S,⁶⁴Cu, ⁷⁶Br, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁵I, or ¹⁷⁷Lu, enzymes, such ashorseradish peroxidase, fluorophores, chromophores, chemiluminescentagents, chelating complexes, dyes, colloidal gold or latex particles.

In one embodiment, particular labels enable detection by emitting adetectable signal of a particular wavelength upon excitation by a laser.Phycobiliproteins, tandem dyes, certain fluorescent proteins, smallchemical molecules, and certain molecules detectable by other means canall be considered labels for flow cytometry analyses. See, e.g., thelabels listed in Handbook of Fluorescent Probes and Research Chemicals,6th Ed., R. P. Haugland, Molecular Probes, Inc., Eugene, Oreg. (1996). Aligand such as an antibody molecule, for example, directly labeled byconjugation with a biliprotein can have as many as 34 associatedchromophores, each with an absorbance and quantum yield roughlycomparable to those of fluorescein. Examples of biliproteins useful inthe certain embodiments described herein are phycocyanin,allophycocyanin (APC), allophycocyanin B, phycoerythrin and preferablyR-phycoerythrin. Phycoerythrin (PE) is among the brightest fluorescentdyes currently available.

As used herein, a “tandem dye” or equivalents thereof refers tonon-naturally occurring molecules that can be formed of a biliproteinand another dye. See, for example, U.S. Pat. No. 4,542,104 and U.S. Pat.No. 5,272,257. Examples of tandem dyes useful in certain embodimentsdescribed herein are phycoerythrocyanin or PC5 (PE-Cy5,phycoerythrin-cyanin 5.1; excitation, 486-580 nm, emission, 660-680 nm)[A. S. Waggoner et al, 1993 Ann. N. Y Acad. Sci., 677:185-193 and U.S.Pat. No. 5,171,846] and ECD (phycoerythrin-texas red; excitation,486-575 nm, emission, 610-635 nm) [U.S. Pat. No. 4,542,104 and U.S. Pat.No. 5,272,257. Other known tandem dyes are PE-Cy5, PE-Cy7, APC-Cy5, andAPC-Cy7 [M. Roederer et al, 1996 Cytometry, 24:191-197]. Thebiliproteins and tandem dyes are commercially available from varioussources including Beckman Coulter, Inc., Miami, Fla., Molecular Probes,Inc., Eugene, Oreg. and Prozyme, Inc., San Leandro, Calif. All of thesefluorescent dyes are commercially available, and their uses known to theart.

Still other labels that can be directly conjugated to the components ofthe certain methods described herein or used with the biliproteins ortandem dyes to add additional numbers of labeled ligands to the methodinclude small molecules that upon excitation emit wavelengths of lessthan 550 nm. Hence, as used herein, a “small molecule” label orequivalents thereof refers to such molecules do not overlap with theemissions of the biliproteins. One example of such a marker isfluorescein isothiocyanate (FITC). Still other labels that can beemployed in this method to provide additional colors are the proteinsknown as the green fluorescent proteins (GFPs) and blue fluorescentproteins (BFPs); also, in certain embodiments, labels that emit uponexcitation by ultraviolet light are useful.

A detectable label can also be an enzyme that interacts with a substrateto produce the detectable signal; or a protein that is detectable byantibody binding or by binding to a suitably labeled ligand. A varietyof enzyme systems operate to reveal a colorimetric signal in an assay,for example, glucose oxidase, horseradish peroxidase (HRP) or alkalinephosphatase (AP), and hexokinase in conjunction with glucose-6-phosphatedehydrogenase that reacts with ATP, glucose, and NAD+ to yield NADH thatis detected as increased absorbance at 340 nm wavelength. Stilladditional labels such as colored latex microparticles (BangsLaboratories, Indiana) whereby a dye is embedded and forms conjugateswith an inhibitor sequence or ligand and provide a visual signalindicative of the presence of the resulting complex can be applicablefor some assays described in certain embodiments. Other label systemsthat can be used include nanoparticles or quantum dots. Thus, any numberof additional, and conventionally employed, labeling systems can beadapted to the methods described herein. One of skill in the artunderstands that selection and/or implementation of a label systeminvolves only routine experimentation. The labels and markers discussedabove can be obtained commercially from known sources.

As used herein, a “solid matrix” or a “solid phase capture medium”refers to any matrix or medium which allows it to be separated from thecell population sample, for example, a physiologically compatible bead.Characteristics of such a matrix or medium include refractive index,size, light scatter intensity, or carrying a fluorescent detector dye toprovide a unique fluorescent signature. Such beads are conventionallyavailable in the art. For example, one subset of solid phase capturemedium includes stable colloidal particles, such as polystyrene beadsranging in size from between about 0.2 to about 5.0 microns in diameter(i.e., colloidal-sized). Such polystyrene substrates or beads cancontain aldehyde and/or sulfate functional groups, such as thecommercially available beads, e.g., from Interfacial DynamicsCorporation, Portland, Oreg.

As used herein, “expression” refers to the production of a material orsubstance as well as the level or amount of production of a material orsubstance. The emerging hES-derived cell populations are assessed byphenotypic markers, and expression patterns are analyzed to determinenot only which factors have a positive or negative influence on thedifferentiation pathway, but also particular cell types. Thus,determining the expression of a specific marker refers to detectingeither the relative or absolute amount of the marker that is expressedor simply detecting the presence or absence of the marker. Statedanother way, if a marker is a protein, polypeptide or fragment orportion thereof, there are various methods of measuring and quantifyingprotein expression for the presence and abundance (levels) of one ormore proteins in a particular cell or tissue. One method is to perform aWestern blot against the marker/protein of interest, whereby cellularlysate is separated on a polyacrylamide gel and then probed with anantibody to the protein of interest. The antibody can either beconjugated to a fluorophore or to horseradish peroxidase for imaging orquantification. Another commonly used method for assaying the amount ofa particular protein in a cell is to fuse a copy of the protein to areporter gene such as Green fluorescent protein (GFP), which can bedirectly imaged using a fluorescent microscope.

In some embodiments, the phrase “does not express” and equivalentsthereof refer to non-detectable expression of a marker or substance. Inother embodiments, the phrase “does not express” and equivalents thereofrefer to marker expression that is detectable but insignificant. Incertain embodiments, insignificant marker expression refers to markerexpression that is detectable by sensitive techniques, such asquantitative polymerase chain reaction, but which is not appreciablydetectable by less sensitive techniques such as immunocytochemistry.

For most markers or targets described herein, the official Human GenomeOrganization (HUGO) gene symbol is provided. Such symbols, which aredeveloped by the HUGO Gene Nomenclature Committee, provide uniqueabbreviations for each of the named human genes and gene products. Thesegene symbols are readily recognized and can easily be associated with acorresponding unique human gene and/or protein sequence by those ofordinary skill in the art.

In accordance with the HUGO designations, the following gene symbols aredefined as follows: GHRL—ghrelin; IAPP—islet amyloid polypeptide;INS—insulin; GCG—glucagon; ISL1—ISL1 transcription factor; PAX6—pairedbox gene 6; PAX4—paired box gene 4; NEUROG3—neurogenin 3 (NGN3);NKX2-2—NKX2 transcription factor related, locus 2 (NKX2.2); NKX6-1—NKX6transcription factor related, locus 1 (NKX6.1); IPF1—insulin promoterfactor 1 (PDX1); ONECUT1—one cut domain, family member 1 (HNF6);HLXB9—homeobox B9 (HB9); TCF2—transcription factor 2, hepatic (HNF1b);FOXA1—forkhead box A1; HGF—hepatocyte growth factor; IGF1—insulin-likegrowth factor 1; POU5F1—POU domain, class 5, transcription factor 1(OCT4); NANOG—Nanog homeobox; SOX2—SRY (sex determining region Y)-box 2;CDH1—cadherin 1, type 1, E-cadherin (ECAD); T—brachyury homolog (BRACH);FGF4—fibroblast growth factor 4; WNT3—wingless-type MMTV integrationsite family, member 3; SOX17—SRY (sex determining region Y)-box 17;GSC—goosecoid; CER1—(cerberus 1, cysteine knot superfamily, homolog(CER); CXCR4—chemokine (C—X—C motif) receptor 4; FGF17—fibroblast growthfactor 17; FOXA2—forkhead box A2; SOX7—SRY (sex determining regionY)-box 7; SOX1—SRY (sex determining region Y)-box 1;AFP—alpha-fetoprotein; SPARC—secreted protein, acidic, cysteine-rich(osteonectin); and THBD—thrombomodulin (TM), NCAM—neural cell adhesionmolecule; SYP—synaptophysin; ZIC1—Zic family member 1;NEF3—neurofilament 3 (NFM); SST—somatostatin; MAFA—v-mafmusculoaponeurotic fibrosarcoma oncogene homolog A; MAFB—v-mafmusculoaponeurotic fibrosarcoma oncogene homolog B; SYP—synaptophysin;CHGA—chromogranin A (parathyroid secretory protein 1).

The following provides the full gene names corresponding to non-HUGOgene symbols as well as other abbreviations that can be used herein:SS—somatostatin (SOM); PP—pancreatic polypeptide; C-peptide—connectingpeptide; Ex4—exendin 4; NIC—nicotinamide andDAPT—N—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester; RA—retinoic acid; RPMI—Roswell Park Memorial Institute medium;CMRL—Connaught Medical Research Labs medium; FBS—fetal bovine serum;NBP10—NCAM binding protein 10; PTF1a—pancreas specific transcriptionfactor 1a. The terms fibroblast growth factor 7 (FGF7) and keratinocytegrowth factor (KGF) are synonymous.

The features and other details of the embodiments described herein,either as steps of certain embodiments or as combinations of parts ofspecific embodiments, will now be more particularly described. It willbe understood that the particular embodiments described herein are shownby way of illustration and not as limitations. The principle features ofthis invention can be employed in various embodiments without departingfrom the scope of the invention.

Human Embryonic Stem Cells and hES-Derived Cell Types

In some embodiments, the differentiation culture conditions andhES-derived cell types described herein are substantially similar tothat described in D'Amour et al. 2006. D'Amour et al. 2006 describe a 5step differentiation protocol which has been adapted and modified inFIG. 1: stage 1 (about 2-4 days) produces definitive endoderm (DE) fromembryonic stem (ES) cells and through a mesendoderm (ME) intermediate,stage 2 (about 3 days) produces primitive gut tube (PG), stage 3 (about3-4 days) produces posterior foregut (PF; or Pdx1-positive foregutendoderm), stage 4 (about 3-4 days) produces pancreatic endoderm (PE; orPDX1/NKX6.1 expressing cells), or pancreatic epithelium and endocrineprecursor cells; and stage 5 produces hormone expressing pancreaticendocrine (EN) cells (about d15 or more). See FIG. 1 for a schematicrepresentation of the 5 stage differentiation protocol. FIG. 1 alsodescribes various markers typical or which characterizes the indicatedcell type. Additional embodiments of differentiation culture conditionsand hES-derived cell types described herein can be found in U.S. patentapplication Ser. No. 11/773,944, entitled METHODS OF PRODUCINGPANCREATIC HORMONES, filed Jul. 5, 2007, the disclosure of which isincorporated herein by reference in its entirety.

A preferred embodiment for deriving these hES-derived cell typesutilizes human embryonic stem cells as the starting material. Suchpluripotent cells can be cells that originate from the morula, embryonicinner cell mass or those obtained from embryonic gonadal ridges. Humanembryonic stem cells can be maintained in culture in a pluripotent statewithout substantial differentiation using methods that are known in theart. Such methods are described, for example, in U.S. Pat. Nos.5,453,357, 5,670,372, 5,690,926 5,843,780, 6,200,806 and 6,251,671 thedisclosures of which are incorporated herein by reference in theirentireties.

In some embodiments, hESCs are maintained on a feeder layer or can bemaintained feed-free. In such processes where a feeder layer isemployed, any feeder layer which allows hESCs to be maintained in apluripotent state can be used. One commonly used feeder layer for thecultivation of human embryonic stem cells is a layer of mousefibroblasts. More recently, human fibroblast feeder layers have beendeveloped for use in the cultivation of hESCs. See U.S. patentapplication Ser. No. 10/486,408 entitled ALTERNATIVE COMPOSITIONS &METHODS FOR THE CULTURE OF STEM CELLS, filed Feb. 6, 2004, thedisclosure of which is incorporated herein by reference in its entirety.Alternative processes permit the maintenance of pluripotent hESC withoutthe use of a feeder layer. Methods of maintaining pluripotent hESCsunder feeder-free conditions have been described in U.S. PatentApplication No. 2003/0175956 and U.S. patent application Ser. No.11/875,057, entitled METHODS AND COMPOSITIONS FOR FEEDER FREEPLURIPOTENT STEM CELL MEDIA CONTAINING HUMAN SERUM, filed Oct. 19, 2007,the disclosures of which are incorporated herein by reference in theirentirety.

The human embryonic stem cells used herein can be maintained in cultureeither with or without serum (“serum-free”). In some embryonic stem cellmaintenance procedures, serum replacement is used. In others, serum freeculture techniques, such as those described in U.S. Patent ApplicationNo. 2003/0190748, the disclosure of which is incorporated herein byreference in its entirety, are used.

In some embodiments, stem cells are maintained in culture in apluripotent state by routine passage until it is desired that they bedifferentiated into lineages of the three germ layers, includingendodermal lineages, further including definitive endoderm, foregutendoderm, PDX1 foregut endoderm, pancreatic epithelium, endocrineprecursor cells and/or pancreatic islet hormone-expressing or endocrinecells. Method of differentiating pluripotent hESCs to these cells typesare described in related applications as indicated above, which areherein incorporated in their entirety; and substantially as described ind'Amour et al. 2005 and 2006, supra.

Monitoring of hES-Derived Cells

The progression of the hES-derived cells described herein (e.g., cellsproduced as a result of Stages or Steps 1-5 as described in d'Amour etal. 2006, supra, can be monitored by determining the expression ofmarkers characteristic of each hES-derived cell type along thedevelopmental pathway. In some embodiments, the identification andcharacterization of a hES-derived cell type is by expression of acertain marker or different expression levels and patterns of more thanone marker. Specifically, the presence or absence, the high or lowexpression, of one or more the marker(s) can typify and identify acell-type. Also, certain markers can have transient expression, wherebythe marker is highly expressed during one stage of development andpoorly expressed in another stage of development. The expression ofcertain markers can be determined by measuring the level at which themarker is present in the cells of the cell culture or cell population ascompared to a standardized or normalized control marker. In suchprocesses, the measurement of marker expression can be qualitative orquantitative. One method of quantitating the expression of markers thatare produced by marker genes is through the use of quantitative PCR(Q-PCR). Methods of performing Q-PCR are well known in the art. Othermethods which are known in the art can also be used to quantitate markergene expression. For example, the expression of a marker gene productcan be detected by using antibodies specific for the marker gene productof interest (e.g., e.g. Western blot, flow cytometry analysis, and thelike). In certain processes, the expression of marker genescharacteristic of hES-derived cells as well as the lack of significantexpression of marker genes characteristic of hES-derived cells. Stillfurther methods for characterizing and identifying hES-derived cellstypes is described in related applications as indicated above, which isherein incorporated in its entirety.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for particular markers listed in this disclosure can beobtained from public databases such as GenBank.

The choice of primers for use in nucleic acid amplification will dependon the marker or target nucleic acid sequence. Primers used in preferredembodiments are generally oligonucleotides, usually deoxyribonucleotidesseveral nucleotides in length, that can be extended in atemplate-specific manner by the polymerase chain reaction. The design ofsuitable primers for amplifying a marker or target nucleic acid iswithin the skill of practitioners in the art. In general, the followingfactors are considered in primer design: a) each individual primer of apair preferably does not self-hybridize; b) the individual pairspreferably do not cross-hybridize; and c) the selected pair must havethe appropriate length and sequence homology in order to anneal to twodistinct regions flanking the nucleic acid segment to be amplified.However, not every nucleotide of the primer must anneal to the templatefor extension to occur. The primer sequence need not reflect the exactsequence of the marker or target nucleic acid. For example, anon-complementary nucleotide fragment can be attached to the 5′ end ofthe primer with the remainder of the primer sequence being complementaryto the target. Alternatively, non-complementary bases can beinterspersed into the primer, provided that the primer sequence hassufficient complementarily with the target for annealing to occur andallow synthesis of a complementary nucleic acid strand.

For a convenient detection of the amplified nucleotide acids resultingfrom PCR or any other nucleic acid amplification reactions describedabove or known in the art, primers can be conjugated to a detectablelabel. Detectable labels suitable for use include, but are not limitedto, any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means. Awide variety of appropriate detectable labels are known in the art,which include luminescent labels, enzymatic or other ligands. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as digoxigenin, β-galactosidase, urease,alkaline phosphatase or peroxidase, avidin/biotin complex.

Amplification probe/primer combinations suitable for use inamplification assays include the following: Insulin (GenBank NM_000207):

primers (SEQ ID NO: 1) CAGCCTTTGTGAACCAACACC;  (SEQ ID NO: 2)CGTTCCCCGCACACTAGGTA;  probe  (SEQ ID NO: 3) CGGCTCACACCTGGTGGAAGCTC. Nkx6.1 (NM_006168): primers  (SEQ ID NO: 4) CTGGAGGGACGCACGC; (SEQ ID NO: 5) TCCCGTCTTTGTCCAACAAAA;  probe  (SEQ ID NO: 6)TGGCCTGTACCCCTCATCAAGGATCC.  Pdx1 (NM_000209):  primers  (SEQ ID NO: 7)TGGCGTTGTTTGTGGCTG; (SEQ ID NO: 8) AGGTCCCAAGGTGGAGTGC;  probe (SEQ ID NO: 9) TGCGCACATCCCTGCCCTCCTAC.  Ngn3 (NM_020999):  primers (SEQ ID NO: 10) TCTCTATTCTTTTGCGCCGG;  (SEQ ID NO: 11)CTTGGACAGTGGGCGCAC;  probe (SEQ ID NO: 12) AGAAAGGATGACGCCTCAACCCTCG. HNF3B (NM_021784):  primers (SEQ ID NO: 13) CCGACTGGAGCAGCTACTATG; (SEQ ID NO: 14) TACGTGTTCATGCCGTTCAT;  probe  (SEQ ID NO: 15)CAGAGCCCGAGGGCTACTCCTCC;  glucagon (NM_002054):  primers (SEQ ID NO: 16) GCTGCCAAGGAATTCATTGC; (SEQ ID NO: 17)CTTCAACAATGGCGACCTCTTC;  probe (SEQ ID NO: 18)TGAAAGGCCGAGGAAGGCGAGATT.  HNF6 (NM_030712):  primers (SEQ ID NO: 19)GCTGGGGTGACCTCAATCTA;  (SEQ ID NO: 20) CAGGAACCTGCATGAGGACT;  probe (SEQ ID NO: 21) AGTTTCAGAGCCATTGGGCGGTG;  HNF4A (NM_000457):  primers (SEQ ID NO: 22) GAGATCCATGGTGTTCAAGGA; (SEQ ID NO: 23)GTCAAGGATGCGTATGGACA;  probe (SEQ ID NO: 24) CTACATTGTCCCTCGGCACTGCC. Sox17 (NM_022454):  primers (SEQ ID NO: 25) CAGCAGAATCCAGACCTGCA; (SEQ ID NO: 26) GTCAGCGCCTTCCACGACT;  probe  (SEQ ID NO: 27)ACGCCGAGTTGAGCAAGATGCTGG.  HIxb9 (NM_005515):  primers  (SEQ ID NO: 28)GCCACCTCGCTCATGCTC; (SEQ ID NO: 29) CCATTTCATCCGCCGGTTC;  probe(SEQ ID NO: 30) CCGAGACCCAGGGAAGATTTGGTTCC.  Nkx2.2 (NM_002509): primers (SEQ ID NO: 31) CGAGGGCCTTCAGTACTCC;  (SEQ ID NO: 32)TTGTCATTGTCCGGTGACTC;  probe  (SEQ ID NO: 33) ACTCAAGCTCCAAGTCCCCGGAG. PTF1a (NM_178161): primers  (SEQ ID NO: 34) GAAGGTCATCATCTGCCATCG(SEQ ID NO: 35) GGCCATAATCAGGGTCGCT.  SST (NM_ 001048): primers (SEQ ID NO: 36) CCCCAGACTCCGTCAGTTTC;  (SEQ ID NO: 37)TCCGTCTGGTTGGGTTCAG;  and  PAX6 (NM_000280):  primers (SEQ ID NO: 38)CCAGAAAGGATGCCTCATAAAGG;  (SEQ ID NO: 39) TCTGCGCGCCCCTAGTTA. Oct4 primers: (SEQ ID NO: 40) TGGGCTCGAGAAGGATGTG  (SEQ ID NO: 41)GCATAGTCGCTGCTTGATCG.  MIXL1 primers (SEQ ID NO: 42)CCGAGTCCAGGATCCAGGTA (SEQ ID NO: 43) CTCTGACGCCGAGACTTGG. GATA4 primers  (SEQ ID NO: 58) CCTCTTGCAATGCGGAAAG (SEQ ID NO: 44)CGGGAGGAAGGCTCTCACT.  FOXA2 primers (SEQ ID NO: 45) GGGAGCGGTGAAGATGGA(SEQ ID NO: 46) TCATGTTGCTCACGGAGGAGTA.  GSC primers  (SEQ ID NO: 47)GAGGAGAAAGTGGAGGTCTGGTT (SEQ ID NO: 48) CTCTGATGAGGACCGCTTCTG. CER primers (SEQ ID NO: 49) ACAGTGCCCTTCAGCCAGACT (SEQ ID NO: 50)ACAACTACTTTTTCACAGCCTTCGT.  AFP primers (SEQ ID NO: 51)GAGAAACCCACTGGAGATGAACA  (SEQ ID NO: 59) CTCATGGCAAAGTTCTTCCAGAA. SOX1 primers (SEQ ID NO: 52) ATGCACCGCTACGACATGG (SEQ ID NO: 53)CTCATGTAGCCCTGCGAGTTG.  ZIC1 primers  (SEQ ID NO: 54)CTGGCTGTGGCAAGGTCTTC (SEQ ID NO: 55) CAGCCCTCAAACTCGCACTT.  NFM primers(SEQ ID NO: 56) ATCGAGGAGCGCCACAAC (SEQ ID NO: 57) TGCTGGATGGTGTCCTGGT. Other primers are available though ABI Taqman including FGF17 (Hs00182599_m1), VWF(Hs00169795_m1), CMKOR1 (Hs00604567_m1), CRIP1(Hs00832816_g1), FOXQ1 (Hs00536425_s1), CALCR(Hs00156229_m1) and CHGA (Hs00154441_m1).

In other embodiments, the expression levels of any marker in hES-derivedcell types or hES-derived cell populations is at least about 4-foldhigher, at least about 6-fold higher, at least about 8-fold higher, atleast about 10-fold higher, at least about 15-fold higher, at leastabout 20-fold higher, at least about 40-fold higher, at least about80-fold higher, at least about 100-fold higher, at least about 150-foldhigher, at least about 200-fold higher, at least about 500-fold higher,at least about 750-fold higher, at least about 1000-fold higher, atleast about 2500-fold higher, at least about 5000-fold higher, at leastabout 7500-fold higher or at least about 10,000-fold higher as comparedto a standardized or normalized control marker.

Purification of hES-Derived Cells

With respect to additional aspects of the embodiments described herein,the hES-derived cell types described herein can be enriched, depleted,isolated, separated, sorted and/or purified as further described in theexamples. As used herein, the terms “enriched” or “purified” or enrichedor purified due to depletion of other known cell populations, indicatethat the cell population has been subject to some selection process sothat the population is enriched and/or purified. Also, the subject cellsare also considered relatively enriched and/or purified, i.e. there issignificantly more of a particular hES-derived cell type population ascompared to another hES-derived type population, or as compared topluripotent undifferentiated hES cells before “enrichment” or“purification”, or as compared to the original or initial cell culture.That said, sometimes enriching or purifying for particular hES-derivedcells types can involve “depleting” or “separating” or “sorting” one ormore known hES-derived cell type from another hES-derived cell type. Forexample, in some embodiments, certain hES-derived cell types areenriched or purified because they do not bind or cross-react to a ligandand/or agent and/or antibody and/or antibody bound to solid orsemi-solid matrix. Such enriched or purified populations can be said tobe “depleted” or “separated” or “sorted” from the hES cell culture. As afurther example, such depleted or separated or sorted hES-cellpopulations can be found or isolated in the flow-through fractions in astandard affinity chromatography method; or stated in another way, thenon-binding fractions. Accordingly, in certain embodiments, it isadvantageous to enrich and purify an hES-derived cell type by depletingthe culture of known or unknown cell types. In this way, the enriched orpurified cell population does not have the bound or attached antibody.Because there is no need to remove the antibody from the purifiedpopulation, the use of the enriched or purified cells for cell therapiesis improved.

The hES-derived cells of the present invention can be produced byemploying methods described in the related application as indicatedabove. For example, methods of expanding definitive endoderm isdescribed in U.S. patent application Ser. No. 11/317,387, entitledEXPANSION OF DEFINITIVE ENDODERM, filed Dec. 22, 2005; U.S. patentapplication Ser. No. 11/860,494, entitled METHODS FOR INCREASINGDEFINITIVE ENDODERM PRODUCTION, filed Sep. 24, 2007; U.S. patentapplication Ser. No. 11/588,693, entitled PDX1-EXPRESSING DORSAL ANDVENTRAL FOREGUT ENDODERM, filed Oct. 27, 2006; U.S. patent applicationSer. No. 11/681,687, entitled ENDOCRINE PRECURSOR CELLS, PANCREATICHORMONE EXPRESSING CELLS AND METHODS OF PRODUCTION, filed Mar. 2, 2007;U.S. patent application Ser. No. 11/773,944 entitled METHODS FORPRODUCING PANCREATIC HORMONES, filed Jul. 5, 2007; and U.S. patentapplication Ser. No. 11/860,494 entitled METHODS OF INCREASINGDEFINITIVE ENDODERM, filed Sep. 24, 2007, which are herein incorporatedin their entirety. One method described herein and in d'Amour et al.2005 and 2006, supra, is directed differentiation of a pluripotent stemcell to different hES-derived cell types, e.g., definitive endoderm andinsulin secreting type cells.

Human ES-derived cells described herein can be enriched, depleted,isolated, separated, sorted and/or purified by using an affinity tagthat is specific for such cells, e.g., an antibody. Examples of affinitytags specific for hESCs or hES-derived cells are polyclonal and/ormonoclonal antibodies, antibody binding fragments, ligands or otherbinding agents that are specific to a marker molecule, such as apolypeptide, that is present on the cell surface of hES-derived cell orpluripotent hESC, but which is not substantially present on other celltypes that would be found in a cell culture produced by the methodsdescribed herein.

Embodiments for enriching, depleting, isolating, separating, sortingand/or purifying include those described herein such as, antibody-coatedmagnetic beads, affinity chromatography and “panning” with antibodyattached to a solid matrix or solid phase capture medium, e.g. plate,column or other convenient and available technique. Techniques providingaccurate separation include flow cytometry methods which are useful formeasuring cell surface and intracellular parameters, as well as shapechange and granularity and for analyses of beads used as antibody- orprobe-linked reagents. Readouts from flow cytometry assays include, butare not limited to, the mean fluorescence associated with individualfluorescent antibody-detected cell surface molecules or cytokines, orthe average fluorescence intensity, the median fluorescence intensity,the variance in fluorescence intensity, or some relationship amongthese. In some aspects of embodiments with analytical steps involvingflow cytometry, minimal parameters or characteristics of the beads arescatter (FS and/or SS) and at least one fluorescent wavelengths. Flowcytometry can be used to quantitate parameters such as the presence ofcell surface proteins or conformational or posttranslationalmodification thereof; intracellular or secreted protein, wherepermeabilization allows antibody (or probe) access, and the like. Flowcytometry methods are known in the art, and described in the following:Flow Cytometry and Cell Storing (Springer Lab Manual), Radbruch, Ed.,Springer Verlag, 2000; Ormerod, Flow Cytometry, Springer Verlag, 1999;Flow Cytometry Protocols (Methods in Molecular Biology, No 91),Jaroszeski and Heller, Eds., Humana Press, 1998; Current Protocols inCytometry, Robinson et al., eds, John Wiley & Sons, New York, N.Y.,2000.

Also, the staining intensity of cells can be monitored by flowcytometry, where lasers detect the quantitative levels of fluorochrome(which is proportional to the amount of cell surface marker bound byspecific reagents, e.g. antibodies). Flow cytometry, or FACS, can alsobe used to separate cell populations based on the intensity of bindingto a specific reagent, as well as other parameters such as cell size andlight scatter. Although the absolute level of staining can differ with aparticular fluorochrome and reagent preparation, the data can benormalized to a control. In order to normalize the distribution to acontrol, each cell is recorded as a data point having a particularintensity of staining. These data points can be displayed according to alog scale, where the unit of measure is arbitrary staining intensity. Inone example, the brightest cells in a population are designated as 4logs more intense than the cells having the lowest level of staining.When displayed in this manner, it is clear that the cells falling in thehighest log of staining intensity are bright or high, while those in thelowest intensity are negative. The “low” staining cells, which fall inthe 2-3 log of staining intensity, can have properties that are uniquefrom the negative and positive cells. In an alternative embodiment, thecontrol utilizes a substrate having a defined density of marker on itssurface, for example a fabricated bead or cell line, which provides thepositive control for intensity. The “low” designation indicates that thelevel of staining is above the brightness of an isotype matched control,but is not as intense as the most brightly staining cells normally foundin the population. The readouts of selected parameters are capable ofbeing read simultaneously, or in sequence during a single analysis, asfor example through the use of fluorescent antibodies to cell surfacemolecules. As an example, these can be tagged with differentfluorochromes, fluorescent bead, tags, e.g. quantum dots, etc., allowinganalysis of up to 4 or more fluorescent colors simultaneously by flowcytometry. For example, a negative designation indicates that the levelof staining is at or below the brightness of an isotype matched negativecontrol; whereas a dim designation indicates that the level of stainingcan be near the level of a negative stain, but can also be brighter thanan isotype matched control.

Identifiers of individual cells, for example different cell types orcell type variants, can be fluorescent, as for example labeling ofdifferent unit cell types with different levels of a fluorescentcompound, and the like as described herein above. In some aspects ofembodiments where two cell types are to be mixed, one is labeled and theother not. In some aspects of embodiments where three or more cell typesare to be included, each cell type is labeled to different levels offluorescence by incubation with different concentrations of a labelingcompound, or for different times. As identifiers of large numbers ofcells, a matrix of fluorescence labeling intensities of two or moredifferent fluorescent colors can be used, such that the number ofdistinct unit cell types that are identified is a number of fluorescentlevels of one color, e.g., carboxyfluorescein succinimidyl ester (CFSE),times the number of fluorescence levels employed of the second color,e.g. tetramethylrhodamine isothiocyanate (TRITC), or the like, times thenumber of levels of a third color, etc. Alternatively, intrinsic lightscattering properties of the different cell types, or characteristics ofthe BioMAPs of the test parameters included in the analysis, can be usedin addition to or in place of fluorescent labels as unit cell typeidentifiers.

Still, other techniques for enriching, depleting, separating, sortingand/or purifying include plug-flow flow cytometry which has thepotential to automate the delivery of small samples from unpressurizedsources at rates compatible with many screening and assay applications,and can allow higher throughput, compatible with high throughputscreening, Edwards et al. (1999) Cytometry 37:156-9. Also, both singlecell multi-parameter and multicell multi-parameter multiplex assays,where input cell types are identified and parameters are read byquantitative imaging and fluorescence and confocal microscopy are usedin the art, see Confocal Microscopy Methods and Protocols (Methods inMolecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. Thesemethods are described in U.S. Pat. No. 5,989,833 issued Nov. 23, 1999.For example, an alternative technique allows for staining of dead cellswhich can be eliminated by selection with dyes such as propidium iodide.However, any technique can be employed which is not unduly detrimentalto the viability of the selected cells.

In another aspect, hES-derived cells can be enriched, depleted,separated, sorted and/or purified using conventional affinity orantibody techniques. For example, the ligand and/or antibody can beconjugated with labels to allow for ease of separation of the particularcell type, e.g. magnetic beads; biotin, which binds with high affinityto avidin or streptavidin; fluorochromes, which can be used with afluorescence activated cell sorter; haptens; and the like. Multi-coloranalyses can be employed with the FACS or in a combination ofimmuno-magnetic separation and flow cytometry. In some embodiments,multi-color analysis is of interest for the separation of cells based onmultiple surface antigens. Fluorochro which find use in a multi-coloranalysis include, but are not limited to, phycobiliproteins, e.g.phycoerythrin and allophycocyanins; fluorescein and Texas red asdescribed herein.

In one embodiment, the ligand, agent, and/or antibodies described hereinare directly or indirectly conjugated to a magnetic reagent, such as asuper-paramagnetic microparticle (microparticle). Direct conjugation toa magnetic particle is achieved by use of various chemical linkinggroups, as known in the art. In some embodiments, the antibody iscoupled to the microparticles through side chain amino or sufhydrylgroups and heterofunctional cross-linking reagents. A large number ofheterofunctional compounds are available for linking to entities. Forexample, at least, 3-(2-pyridyidithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle can be used. An example ofa magnetic separation device is described in WO 90/07380,PCT/US96/00953, and EP 438,520, incorporated herein by reference in itsentirety. The purified cell population can be collected in anyappropriate medium.

In other embodiments, antibodies are indirectly coupled to the magneticparticles, e.g., the antibody is directly conjugated to a hapten, andhapten-specific second stage antibodies are conjugated to the particles.Suitable haptens include, but are not limited to, digoxin, digoxigenin,FITC, dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods forconjugation of the hapten to a protein are known in the art, and kitsfor such conjugations are commercially available.

In other embodiments, the antibody is coupled to a solid matrix orcapture medium and added to a cell sample. For example, a capture mediumincludes a polystyrene bead, or a polystyrene bead which has anaminodextran coating over its peripheral surface and/or acolloidal-metal coating. Preferably an aminodextran coating iscovalently bonded to the core substrate by covalent bonds between thefree amino groups of the aminodextran and the amine-reactive functionalgroups of the polystyrene substrate and further by cross-linking with anagent such as glutaraldehyde. A variety of aminodextran beads aredescribed in U.S. Pat. Nos. 6,074,884; 5,945,293; and 5,658,741.Aminodextran-coated monodispersed colloidal dispersions of magneticferrite [U.S. Pat. No. 5,240,640], metal [U.S. Pat. No. 5,248,772],polystyrene [U.S. Pat. No. 5,466,609; U.S. Pat. No. 5,707,877; U.S. Pat.No. 5,639,620; U.S. Pat. No. 5,776,706], and polystyrene-metal [U.S.Pat. No. 5,552,086; U.S. Pat. No. 5,527,713] particles can also beemployed as formed bodies according to particular embodiments describedherein. The disclosures of these patents are herein incorporated byreference in their entirety.

In other embodiments, another type of bead contains the above-describedcoated substrate with a layer of colloidal-sized metallic solidoverlaying the aminodextran coating. Preferably this layer is uniformlydispersed over the dispersed surface of the aminodextran layer. Thecolloidal metal useful in forming the coated substrate is generallydescribed as a metal which can be reduced from the ionic state to themetal(0) state by the aminodextran coating, or a metal which can formmetal ions or metal ion complexes which have a reduction potential ofabout +0.7 volts or higher. In certain embodiments the metal ionsemployed include: Ag(I), Au(III), Pd(II), Pt(II), Rh(III), Ir(III),Ru(II), and Os(II). In a preferred embodiment, metal ions for such useare colloidal gold(III) and colloidal silver(I). Specifically,gold/silver colloid coated polystyrene-aminodextran beads, theirpreparation, characterization and use in analyses of subpopulations ofwhite blood cells in whole blood have been described. See, e.g., U.S.Pat. No. 5,248,772; U.S. Pat. No. 5,552,086; U.S. Pat. No. 5,945,293;and O. Siiman and A. Burshteyn, 2000 J. Phys. Chem., 104:9795-9810; andO. Siiman et al, 2000 Cytometry, 41:298-307, the disclosure of which isherein incorporated by reference in its entirety. Still, an alternativeto this coated bead employs carboxy-functionalized polystyrene particlesas the core substrate, coated with aminodextran by EDAC coupling asdescribed in U.S. Pat. No. 5,639,620, the disclosure of which is hereinincorporated by reference in its entirety.

The capture medium can have bound thereto multiple ligands or multiplecompeting analytes. Each ligand bound to the capture medium is capableof binding to a soluble analyte or binding to an antibody that is itselfcapable of binding to the soluble analyte. Each competing analyte boundto the capture medium is capable of binding to a ligand (e.g., anantibody) that is capable of binding to the soluble analyte (whetherlabeled or unlabeled). Such ligands or competing analytes are associatedor immobilized on the capture medium by conventional methods. Forexample, ligands or analytes such as antibodies, antigens, or linkers(e.g. Streptavidin, Protein A) can be attached to beads depending uponformat of the analyte assay (competitive, immune-complex or sandwich) asdescribed below. The beads can also be associated with detectablelabels, preferably fluorescent labels, such as discussed above.

Beads can be fluorescent or non-fluorescent, can be of different sizesor different fluorescent intensities, or both, for differentiation ofmultiple analytes. If using fluorescent intensity for labeling beads, itis preferred that the fluorescence emission should be unique for eachpopulation directed to a different analyte. Bead populations ofdifferent intensity are preferably resolvable if fluorescence of thebead is used as the only detectable label for discriminating among thesoluble analyte and cellular target. Alternatively, if the size of thebead populations is used as the sole detectable label for discriminationamong the soluble analyte and cellular target, each bead population musthave a different forward scatter (FS) or side scatter (SS) than the cellpopulation of interest in the assay.

In one embodiment the bead is from 0.05 to 20 microns in diameter. Inanother embodiment, the bead is from 5 to 7 microns. In still anotherembodiment, the capture medium is greater than 1 μM in size. Mixtures ofa variety of sizes of beads can also be employed, particularly wherethere are more than one soluble analyte to be detected. Generally, beadsize impacts the sensitivity range of the assay, because smaller beadsbind less antibody. Therefore, in one embodiment, in which highsensitivity is required, a smaller number of larger beads is desirablefor the assays. In the presence of large numbers of soluble analytes, ahigher number of beads (both large and small) can be employed in thesemethods. For use in some embodiments of, the capture medium or bead islarger than the soluble analyte to be detected. The relative volumes ofthe bead used in the sample container of the methods described hereinare dependent upon bead concentration, analyte detection limits, and thecellular marker or target, and sample size.

In preferred embodiments, the antibody is capable of binding to at leastone marker or target on a cell. The amount of the antibody necessary tobind a particular cell subset is empirically determined by performing atest separation and analysis. The cells and antibody are incubated for aperiod of time sufficient for complexes to form, usually at least about5 min, more usually at least about 10 min, and usually not more thanthirty (30) minutes to one hour; time can also be a variable of theincubation temperature.

In still another embodiment, hES-derived cells can be enriched,depleted, separated, sorted, isolated and/or purified using techniquesfor sample purification employing aspects of typical immunity complexassays. In an aspect of this embodiment, a sample, such as a sample ofan hES-derived cell population, is introduced into a container, such asany tissue culture container. A known concentration of a first solubleligand capable of binding to a single cellular target is added to thesample. This first ligand is desirably associated with a firstdetectable label. Multiple of the first ligands may bind to the cell.For example, a known concentration of a first soluble antibody, which iscapable of binding to a single cellular target, is added to ahES-derived cell population in a tissue culture container. Afterincubating from about 5 minutes to about 30 minutes, preferably up to 60minutes, at a temperature of under 37° C. or 4° C., a first complex isformed which includes the cellular target bound to the first labeledligand. Various optional washing steps can be employed after theaddition of the components, depending upon required assay sensitivity.The final step of this method is a simultaneous analysis of the sampletreated as described above, by taking the hES-derived cell populationsamples containing these complexes and discriminating between thecomplex comprising the cellular target bound to the first labeled ligandand/or antibody and any of the sample that did not bind to the solubleligand. The amount of first complex detected is proportional to theamount of the particular cell type having the single cellular targetpresent in the hES-derived cell population.

As with the above assay, some embodiments described herein provide formeasurement of more than one cell type, more than one cellular target ona cell type, or more than one soluble analyte by selecting from amongany number of soluble ligands, detectable labels, and solid phasecapture media on which is immobilized different ligands or competinganalytes. Methods suitable for performing the analysis step includeimage analysis and, preferably, flow cytometric analysis. A flowcytometric analysis is conducted by employing a gating strategyappropriate to the sample type. For example, the complexes containingthe beads are gated separately from the complex of the ligand-labeledcells based on light scatter and fluorescence intensity. Thereafter, ifmore than one fluorescent label is present on the cell target or thebead, the strategy can provide separate compensation for eachfluorophore. Similarly other cell parameters, such as differential andintracellular antigens or other targets can also be measured during thisanalysis.

Some embodiments also include a number of optional steps. For example,where increased sensitivity of the assays are desirable, washing stepswith buffer, or diluent can be introduced into the methods. Generally,such washing steps can be introduced after the incubation of the samplewith the capture medium to eliminate materials not bound to the capturemedium. Alternatively, such washing steps can follow incubation withsoluble ligand to eliminate uncomplexed materials.

Additionally, embodiments described herein encompass various media to beused and including commercially available media such as Dulbecco'sModified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS),Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's modifiedDulbecco's medium (IMDM), phosphate buffered saline (PBS) with 5 mMEDTA, etc., frequently supplemented with fetal calf serum (FCS), bovineserum albumin (BSA), human serum albumin (HSA), StemPro®hESC SFM(Novocell defined media licensed to and sold by Invitrogen) etc.

Using the methods described herein, cell populations or cell culturescan be enriched in cell content by at least about 2- to about 1000-foldas compared to untreated cell populations or cell cultures. In someembodiments, hES-derived cells can be enriched by at least about 5- toabout 500-fold as compared to untreated cell populations or cellcultures. In other embodiments, hES-derived cells can be enriched fromat least about 10- to about 200-fold as compared to untreated cellpopulations or cell cultures. In still other embodiments, hES-derivedcells can be enriched from at least about 20- to about 100-fold ascompared to untreated cell populations or cell cultures. In yet otherembodiments, endocrine precursor cells can be enriched from at leastabout 40- to about 80-fold as compared to untreated cell populations orcell cultures. In certain embodiments, hES-derived cells can be enrichedfrom at least about 2- to about 20-fold as compared to untreated cellpopulations or cell cultures.

Kits

For convenience, in some embodiments the conventional reagents for highthroughput assays or other diagnostic assays can be provided in the formof kits. In some embodiments, a kit is provided for performance of theabove-described methods. In preferred embodiments, such kits areemployed for performing the diagnostic methods described herein and/ormonitoring therapy. In other embodiments, such kits are assembled forresearch purposes also. Thus, in preferred embodiments a kit containsthe components taught above, e.g., at least one soluble ligand thatbinds a cellular target in the sample; at least one soluble ligand thatbinds a soluble analyte in the sample or at least one competing solubleanalyte (preferably labeled); and a solid phase capture medium thatbinds directly to the soluble analyte, indirectly to the solubleanalyte, or to the soluble ligand that binds to the soluble analyte. Thekits also include instructions for performing the particular assay,various diluents and buffers, and signal-generating reagents, such asfluorophores, enzyme substrates, cofactors and chromogens. Othercomponents can include indicator charts for colorimetric comparisons,disposable gloves, decontamination instructions, applicator sticks orcontainers, and a sample preparator cup.

In one embodiment, a kit useful for the performance of theabove-described immuno assays include, as a component, a solid matrix ora solid phase capture medium associated with multiple first ligands thatbind the cellular marker or target and is associated with a firstdetectable label. The kit further comprises a second, third and/orfourth ligand that is capable of binding to a second, third and/orfourth cellular marker or target and or soluble analyze. The second,third and/or fourth ligands are associated with a second, third and/orfourth detectable label.

In another embodiment, a kit for performing another of the competitiveinhibition assays described above, contains a first ligand associatedwith a first label. Multiple of the first ligands are capable of bindingto a single cellular target. Another component is a competing analyteassociated with a second label. Still another component is the solidphase capture medium on which are immobilized multiple of ligands orantibodies capable of binding to the soluble analyte (either competingsoluble analyte or soluble analyte naturally occurring in the sample).

In yet another embodiment, a kit for performing the immune complex assayincludes a first ligand capable of binding to a first cellular targetand providing a first detectable signal; a second ligand capable ofbinding to the soluble analyte and providing a second detectable signal;a third ligand capable of binding to the same soluble analyte; a solidphase capture medium on which is immobilized multiple fourth ligands,the fourth ligands capable of binding to the third ligands.

Such kits are useful for evaluating hES populations and/or hES-derivedcell population samples for purposes of enriching, isolating, depleting,separating, sorting, purifying and/or determining levels of certain celltypes or bound components or soluble antigens or analytes thereof. Sucha diagnostic kit contains the dyes, ligands, capture medium, and othercomponents of the embodiments described herein. Such kits also containlabels, exemplified above, pre-attached to the other components of thespecific assay to be performed, or provided separately for attachment toa selected component, e.g., a substrate. Alternatively, such kits cancontain a simple mixture of such compositions or means for preparing asimple mixture.

Having generally described certain embodiments, a further understandingcan be obtained by reference to certain specific examples which areprovided herein for purposes of illustration only, and are not intendedto be limiting.

Example 1 Human ES-Derived Cell Specific CD Antigens

To enable isolation and purification of hES-derived cells, such as humandefinitive endoderm cells, gut endoderm cells, pancreatic epithelialcells and/or pancreatic endoderm type cells, or endocrine cells fromhuman embryonic stem cells (hESCs), a high-throughput flow cytometrybased screen of antibodies was performed to determine novel cell surfaceantigens on the hES-derived cells. The antibodies were obtained from 3different companies including BD Pharmingen™ (La Jolla, Calif.),Biolegend (San Diego, Calif.), and Millipore Corporation (Temecula,Calif.). The antibodies obtained from BD Pharmingen were a generousgift. The antibodies are set forth in Table 1 below.

TABLE 1 ANTIBODIES Antibody CyT203 CyT203 CyT49 Isotype Antigen CloneCompany Catalog # stage 2 stage 5 stage 4 Ms IgG1, k CD1d CD1d42 BD550254 rare − − Ms IgG1, k CD6 M-T605 BD 555356 rare − − Ms IgG1, k CD9M-L13 BD 555370 + + + Ms IgG1, k CD10 HI10a BD 555373 + + + Ms IgG1, kCD13 WM15 BD 555393 + + + Ms IgM, k CD15 HI98 BD 555400 + + + Ms IgG2a,k CD24 ML5 BD 555426 + + + Ms IgG1, k CD25 M-A251 BD 555430 rare rare −Ms IgG1, k CD26 M-A261 BD 555435 + + + Ms IgG1, k CD27 M-T271 BD 555439− rare − Ms IgG2a, k CD29 HUTS-21 BD 556048 + + + Ms IgG1, k CD30 BerH8BD 555827 + − − Ms IgG1, k CD31 WM59 BD 555444 + − − Ms IgG2b, k CD32FLI8.26 BD 555447 − − rare Ms IgG1, k CD34 581 BD 555820 + + + Ms IgM, kCD36 CB38 (NL07) BD 555453 rare − − Ms IgG1, k CD40 5C3 BD 555587 + − −Ms IgG2b, k CD44 G44-26 BD 555476 + − − Ms IgG2a, k CD45RO UCHL1 BD555491 rare − − Ms IgG2a, k CD46 E4.3 BD 555948 + + + Ms IgG1, k CD47B6H12 BD 556044 + + + Ms IgM, k CD48 T00dc145 BD 555758 rare − − MsIgG1, k CD49a SR84 BD 559594 + + + Ms IgG1, k CD49b AK-7 BD 555497 + + +Ms IgG2a, k CD49b 12F1-H6 BD 555668 + + + Ms IgG1, k CD49c C3 II.1 BD556024 + + + Ms IgG1, k CD49e IIA1 BD 555615 + + + Ms IgG1, k CD49e VC5BD 555651 + + + Ms IgG2a, k CD49f GoH3 BD 555734 rare − − Ms IgG2b, kCD50 T00dc41 BD 555957 + − − Ms IgG1, k CD54 HA58 BD 555510 + − + MsIgG2a, k CD55 IA10 BD 555691 + + + Ms IgG2b, k CD56 NCAM16.2 BD559043 + + + Ms IgM, k CD57 NK-1 BD 555618 + + + Ms IgG2a, k CD58 1C3 BD555919 + + + Ms IgG2a, k CD59 p282 (H19) BD 555761 + + + Ms IgG1, k CD61VI-PL2 BD 555752 rare − rare Ms IgG1, k CD62E 68-5H11 BD 555648 − − rareMs IgG1, k CD63 H5C6 BD 556019 + + + Ms IgG1, k CD66 B6.2/CD66 BD551355 + + + Ms IgM, k CD66b G10F5 BD 555723 rare − − Ms IgG2a, k CD71M-A712 BD 555534 + + + Ms IgG1, k CD73 AD2 BD 550256 − + + Ms IgM, kCD77 5B5 BD 551352 rare − − Ms IgG1, k CD79b CB3.1 BD 555678 rare rare −Ms IgG1, k CD81 JS-81 BD 555675 + + + Ms IgG1, k CD83 HB15e BD 556854rare rare rare Ms IgG2b, k CD85 GHI/75 BD 555941 rare − − Ms IgG1, kCD90 5E10 BD 555593 + + + Ms IgG1, k CD91 A2MR-alpha 2 BD 550495 + + +Ms IgG2b CD93 R139 Millipore MAB4314 rare rare nd Ms IgG1, k CD95 DX2 BD555671 − + + Ms IgG1, k CD98 UM7F8 BD 556074 + + + Ms IgG2a, k CD99T00dc12 BD 555687 + + + Ms IgM, k CD99R HIT4 BD 555817 + − + Ms IgG1, lCD104 450-9D BD 555721 + − − Ms IgG1, k CD105 43A3 Biolegend 323201 rarerare nd Ms IgG1, k CD106 51-10C9 BD 555645 + − − Ms IgG2a, k CD108 KS-2BD 552423 + − − Ms IgG1, k CD112 (PRR2) R2.525 BD 551056 − + − Ms IgG1,κ CD116 4H1 Biolegend 305901 + − − Ms IgG1, k CD117 YB5.B8 BD 555713 + −− Ms IgG1, k CD123 9F5 BD 555642 − − rare Ms IgG1, k CD130 AM64 BD555756 + + + Ms IgG1, k CD134 ACT35 BD 555836 rare − − Ms IgG1, k CD138Mi15 BD 551902 rare − − Ms IgG2a, k CD140a alpha R1 BD 556001 + + − MsIgG2a, k CD140b 28D4 BD 558820 + + + Ms IgG1, k CD141 1A4 BD559780 + + + Ms IgG1, k CD142 HTF-1 BD 550252 + + + Ms IgG1, k CD146P1H12 BD 550314 + + + Ms IgG1, k CD147 HIM6 BD 555961 + + + Ms IgG1, kCD150 A12 BD 559591 − rare − Ms IgG1, k CD151 14A2.H1 BD 556056 + + + MsIgG1, k CD153 D2-1173 BD 550052 rare − − Ms IgM, k CD158a HP-3E4 BD556061 rare − − Ms IgG2a, k CD164 N6B6 BD 551296 + + + Ms IgG1, k CD165SN2 BD 556050 + + + Ms IgG1, k CD166 3A6 BD 559260 + + + Ms IgG1, κCD172a SE5A5 Biolegend 323801 + + + Ms IgG1, k CD172b B4B6 BD 552024 −rare − Ms IgG1, k CD177 MEM-166 BD 551899 + + + Ms IgG2a, k CD184 12G5BD 555972 + + + Ms IgG1, k CD200 MRC OX-104 BD 552023 + + + Rat IgG1, kCD201 RCR-252 BD 552500 − rare − Ms IgG2b, k CD209 DCN46 BD 551186 rarerare − Ms IgG1, κ CD218a H44 Biolegend 313803 rare − − Ms IgG1, k CD2203B6/IR BD 559954 + + + Ms IgG1, k CD221 3B7 BD 556000 + + + Ms IgG1, κCD222 MEM-238 Biolegend 315901 + + + Ms IgG1, k CD227 HMPV BD555925 + + + Ms IgG1, κ CD261 DJR1 Biolegend 307201 − rare rare Ms IgG1,κ CD266 ITEM-1 Biolegend 314001 + + + Ms IgG2b, κ CD318 CUB1 Biolegend324001 + + + Ms IgG1, κ CD334 4FR6D3 Biolegend 324305 + n.d. n.d. MsIgG1, κ CD340 24D2 Biolegend 324401 + + + Ms IgG2b, k ABCG2 5D3 BD552823 + + − Ms IgM, k αβ TCR T10B9.1A-31 BD 555546 rare − − Ms IgM, kβ2-microglobulin T00dc99 BD 555550 + − − Ms IgG3, k Blood Group ANaM87-1F6 BD 550806 + + − Ms IgM, k CMRF44 CMRF44 BD 551055 − + − RatIgM, k CLA HECA-452 BD 555946 − rare − Ms IgG2b, k EGF Receptor EGFR1 BD555996 + + + Ms IgG1, k F11 Receptor M.AB.F11 BD 552147 + + + Ms IgG1, kfMLP receptor 5F1 BD 556015 − rare − Ms IgG1, k γδ□TCR B1 BD 555715 −rare − Ms IgG1, k HLA-A, B, C G46-2.6 BD 555551 + + + Ms IgG2b, k HLA-A2BB7.2 BD 551230 + − − Ms IgG1, k mu -Calpain B27D8 BD 550935 − − + MsIgG1, k NGF receptor C40-1457 BD 557194 + − + Ms IgG1, k Siglec-6E20-1232 BD 550908 + − +

hESC culture preparation: Human embryonic stem cells (hESCs; CyT49 andCyT203 lines) were differentiated in vitro to either stage 2 for about5-6 days or to stage 4 or 5 for about 13-22 days substantially asdescribed in D'Amour et al. 2006 Nat Biotechnol. 24(11):1392-401 andU.S. Patent Application Publication Number 2007/0154984, the disclosuresof which are incorporated herein by reference in their entireties.Briefly, undifferentiated human embryonic stem (hES) cells weremaintained on mouse embryo fibroblast feeder layers (Millipore, formerlyChemicon or Specialty Media) or on human serum coated plates (ValleyBiomedical) in DMEM/F12 (Mediatech) supplemented with 20% KnockOut serumreplacement (Invitrogen/Gibco), 1 mM nonessential amino acids(Invitrogen/Gibco), Glutamax (Invitrogen/Gibco), penicillin/streptomycin(Invitrogen/Gibco), 0.55 mM 2-mercaptoethanol (Invitrogen/Gibco) and 4ng/ml to 20 ng/mL recombinant human FGF2 (R&D Systems). Activin A (R&DSystems) was added to the growth culture medium at 10-25 ng/ml to helpmaintain undifferentiated growth. Cultures were either manuallypassaged, passaged using 5 ug/mL dispase (Invitrogen/Gibco), or passagedusing Accutase (Innovative Cell Technologies #AT104) at 1:4-1:15 splitratios every 3-7 days. Before initiating differentiation, hES cells weregiven a brief wash in PBS+/+ (Mediatech).

Differentiation: Human ES cells were differentiated in RPMI (Mediatech)supplemented with Glutamax, penicillin/streptomycin, 100 ng/mL activin Aand varying concentrations of defined FBS (HyClone). Additionally, 0.1%BSA (Invitrogen/Gibco) and 25 ng/mL-75 ng/ml Wnt3a was added on thefirst day (d0) of differentiation. In most differentiation experimentsFBS concentrations were 0% for the first 24 h, 0.2% for the second 24 h,and 0.2% for the third 24h, when a third stage 1 day was used.Recombinant human activin A and Wnt3a were purchased from R&D Systems.

Subsequently, at stage 2, cells were briefly washed in PBS+/+ and thendifferentiated in RPMI supplemented with 2% FBS, Glutamax,penicillin/streptomycin, and 25 ng-50 ng/mL KGF (R&D Systems) for 3days. In some experiments 5 uM SB431542 (Sigma Aldrich, Inc.) was addedduring the first day of stage 2.

Subsequently (stage 3), cells were differentiated in DMEM (Hyclone)supplemented with Glutamax, penicillin/stretopmycin, and0.5×B27-supplement (Invitrogen/Gibco). Media was additionallysupplemented with either 1 uM to 2 uM Retinoic acid (Sigma) and 0.25 nMkaad-cyclopamine (Toronto Research Chemicals) for 1 to 3 days. In othercases noggin was added at 50 ng/mL (R&D systems) along with the retinoicacid and kaad-cyclopamine. Alternatively, 0.2 uM to 0.5 uM retinoic acidand 0.25 nm Kaad-cyclopamine was added to the media for one day. In someexperiments no retinoic acid or kaad-cyclopmaine was added.

At stage 4, after withdrawal of retinoic acid or in cases where noretinoic acid was added, noggin at 30-100 ng/mL was added to the mediafor 1-9 days, and in some experiments FGF10 at 25 ng/mL was also added.

For stage 5 differentiation was continued in either CMRL(Invitrogen/Gibco) or DMEM (Hyclone) supplemented with Glutamax,penicillin/stretopmycin, and 0.5×B27-supplement (Invitrogen). In someexperiments media was also supplemented with 0.5% human serum (ValleyBiomedical) during stage 5.

Cells were collected at stages 2 (endodermal lineage screen), late stage4/early stage 5 (pancreatic endoderm and endocrine screen) and stage 5(pancreatic endoderm and endocrine screen). Cells were briefly washed inPBS. Using either TrypLE (Invitrogen #12563-011) or Accutase (InnovativeCell Technologies #AT104) at 37° C. cells were enzymatically dissociatedto single cell suspension. 3 to 10% FBS/PBS was added and the suspensionwas passed through a 40-100 um filter and pelleted. The cells werewashed in 3% FBS/PBS (buffer). Cells were pelleted and resuspended as asingle cell suspension in buffer to block nonspecific antibody binding.Cells were incubated with the antibodies as described in Table 1 in avolume of 100 ul buffer containing 0.3-1×10e6 cells in a 96 well platewith 2 ul of antibody for 30-60 minutes at 4 degrees Celsius. Cells werepelleted and then washed 2 times in 2× volume buffer. Cells were thenincubated in a volume of 100 ul buffer containing 0.3-1×10e6 cells in a96 well plate with 2.5 ul of the appropriate fluorochrome conjugatedsecondary antibody for 30-60 minutes at 4 degrees Celsius. Cells werepelleted and then washed 2 times in 2× volume buffer. 0.3-1×10e6 cellswere then resuspended in 200 ul-350 ul volume of 1% paraformaldehyde(Alfa Aesar) and placed in microdilution tubes for flow cytometryanalysis.

Flow cytometry analysis was performed on a FACSCalibur™ (BectonDickinson), according to the manufacturer's instructions and analyzedusing FlowJo flow cytometry analysis software (Tree Star, Inc.). Eachsample containing a cell sample and contacting each of the aboveantibodies in Table 1 was analyzed. Cells were gated for intact cellsusing the parameters of forward and side scatter, and subsequentlyintact cells were analyzed for fluorescence intensity. In some cases,additional gating of intact cells was performed to separate cells intosub-populations on the parameters of forward and side scatter. Thoseantibodies with fluorescent intensity levels at least 3× above thestandard control were characterized as positive, represented by a “+”sign in Table 1. Those antibodies with fluorescent intensity levels atleast 3× below the standard control were characterized as negative,represented by a “−” sign in Table 1. Finally, those antibodies whichstained <=0.5-1% of the intact cells were characterized as rare asstated in Table 1. Some samples having fluorescent intensity levels atleast 3× above the standard controls were characterized further, and atleast those samples with anti-CD30, CD49a, CD49e, CD55, CD57, CD98,CD99, CD142, CD165, CD200, CD318, CD334, and CD340, are described indetail below in Examples 2 through 6.

Based on the data presented in Table 1, any of the antibodies thatresulted in positive or rare staining could be used for positive ornegative immuno-selection of hES-derived endoderm populations at variousstages of differentiation.

Example 2 Purification of Gut Endoderm Cells Using CD30, CD49A, CD49E,CD55, CD99, CD165, and CD334

To further characterize some of the antibodies described in Example1/Table 1, cells were co-stained with both the anti-surface markerantibody and an antibody to a known marker of gut endoderm, thetranscription factor FOXA2. Briefly, CyT203 hES cells weredifferentiated substantially as described in Example 1. On day 6differentiated cells were collected and enzymatically digested to asingle cell suspension, resuspended in buffer (3% FBS/PBS), and stainedwith primary antibodies to cell surface markers as described in Example1 except that in some cases staining was performed in tubes andpost-antibody washes with buffer were typically performed 1-2× with a10× volume of buffer. Cells were incubated in a 1:10 dilution of thefollowing fluorochrome-conjugated or unconjugated antibodies, some ofwhich are also listed in Table 1: anti-CD55−PE (BD Biosciences #555694),CD49a (BD Biosciences #559594), CD49e−PE (BD Biosciences #555617),CD334−PE (Biolegend #324305), CD30 (BD Biosciences #555827), CD99 (BDBiosciences #555687), and CD165 (BD Biosciences #556050).

Following incubation with primary and appropriate secondary antibodies,the cells were fixed in 4% paraformaldehyde for 30 minutes and thenwashed and stored in buffer. For intracellular or intranuclear staining,cells were permeabilized for 30 minutes on ice in PBS containing 5%normal donkey serum (Jackson Immunoresearch) and 0.2% Triton X-100 andthen washed in buffer. Cells were then incubated overnight at 4 degreesCelsius in PBS containing 5% normal donkey serum and 0.1% Triton X-100(blocking buffer) and a 1:100 dilution of anti-FOXA2 antibody (SantaCruz #6554). Cells were then washed in buffer and subsequently incubatedin blocking buffer containing the appropriate secondary antibody for 60minutes at 4 degrees Celsius. Cells were washed 2× in buffer and thenresuspended in buffer or 1% paraformaldehyde for flow cytometryanalysis. Flow cytometry analysis was performed as described in Example1.

Flow cytometry analyses of either anti-CD30, CD49a or CD55 co-stainingwith anti-FOXA2 are shown in Table 2. 38.70% CD30−FOXA2+ cells and atotal of 44.87% FOXA2+ cells were present in the cell population. CD30−cells comprised 86.25% of the total FOXA2+ cells, making CD30 a usefulmarker for negative selection of gut endoderm. 28.91% CD49a(low)FOXA2+cells and a total of 39.29% FOXA2+ cells were present in the population.CD49a(low) cells comprised 73.58% of the total FOXA2+ cells, makingCD49a a useful marker for negative selection of gut endoderm. 26.50%CD55(low)FOXA2+ cells and a total of 37.50% FOXA2+ cells were present inthe population. CD55− cells comprised 70.67% of the total FOXA2+ cells,making CD55 useful for negative selection of gut endoderm.

TABLE 2 CD30, CD49A OR CD55 NEGATIVE IMMUNO- SELECTION FOR FOXA2+ GUTENDODERM CD30 % of total CD30− CD30+ CD30+ CD30− Total Total FOXA2+cells FOXA2+ FOXA2+ FOXA2− FOXA2− FOXA2+ FOXA2− that are CD30− 38.70%6.17% 28.80% 26.30% 44.87% 55.10% 86.25% CD49a % of total CD49a(low)CD49a(high) CD49a(high) CD49a(low) Total Total FOXA2+ cells FOXA2+FOXA2+ FOXA2− FOXA2− FOXA2+ FOXA2− that are CD49a(low) 28.91% 10.38%44.21% 16.38% 39.29% 60.59% 73.58% CD55 % of total CD55(low) CD55(high)CD55(high) CD55(low) Total Total FOXA2+ cells FOXA2+ FOXA2+ FOXA2−FOXA2− FOXA2+ FOXA2− that are CD55(low) 26.50% 11.00% 43.50% 19.10%37.50% 62.60% 70.67%

Flow cytometry analyses of either anti-CD49e, CD99, CD165, or CD334co-staining with anti-FOXA2 are shown in Table 3. About 30.00%CD49e(high)FOXA2+ cells and a total of 39.47% FOXA2+ cells were presentin the cell population. CD49e(high) cells comprised 76.01% of the totalFOXA2+ cells, and hence CD49e can be utilized for positiveimmuno-selection of gut endoderm. Similarly, CD99(high) cells comprised92.72% and CD165+ cells comprised 99.12% of the total FOXA2+ cells. CD99or CD165 can therefore be utilized for positive immuno-selection of gutendoderm as well. CD334+ cells comprised 28.88% of the total FOXA2+cells and 3.6% of the total FOXA2− cells. Hence, CD334 can also be usedfor positive immuno-selection of gut endoderm.

TABLE 3 CD49e, CD99, CD165, or CD334 positive immuno-selection forFOXA2+ gut endoderm CD49e % of total CD49e(low) CD49e(high) CD49e(high)CD49e(low) Total Total FOXA2+ cells FOXA2+ FOXA2+ FOXA2− FOXA2− FOXA2+FOXA2− that are CD49e(high) 9.47% 30.00% 19.10% 41.50% 39.47% 60.60%76.01% CD165 % of total CD165− CD165+ CD165+ CD165− Total Total FOXA2+cells FOXA2+ FOXA2+ FOXA2− FOXA2− FOXA2+ FOXA2− that are CD165+ 0.37%41.70% 40.60% 17.30% 42.07% 57.90% 99.12% CD334 % of total CD334− CD334+CD334+ CD334− Total Total FOXA2+ cells FOXA2+ FOXA2+ FOXA2− FOXA2−FOXA2+ FOXA2− that are CD334+ 29.80% 12.10% 2.10% 56.00% 41.90% 58.10%28.88% CD99 % of total CD99− CD99− CD99(low) CD99(high) CD99(high)CD99(low) Total Total FOXA2+ cells FOXA2− FOXA2+ FOXA2+ FOXA2+ FOXA2−FOXA2− FOXA2+ FOXA2− that are CD99(high) 14.16% 0.74% 2.26% 38.20%16.93% 27.70% 41.20% 58.79% 92.72%

Example 3 Purification of PDX1 Endoderm Cells Using CD55, CD57, CD98,and CD142

To further characterize some of the antibodies described in Example1/Table 1, cells were co-stained with both the anti-surface markerantibody (Table 1) and an antibody to the transcription factor PDX1, amarker of posterior foregut endoderm and pancreatic endoderm. CyT49 hEScells were differentiated substantially as described in Example 1.Differentiated cells were then collected on day 14 and processed forsurface antibody staining as described in Examples 1 and 2 using a 1:10dilution of the following fluorochrome conjugated antibodies: CD55−PE(BD Biosciences #555694), CD55−APC (BD Biosciences #555696), CD57−FITC(BD Biosciences #555619), CD98−FITC (BD Biosciences #556076), CD98−PE(BD Biosciences #556077), CD142−PE (BD Biosciences #550312). Cells werethen processed for intracellular or intranuclear staining substantiallyas described in Example 2 except that post primary antibody and postsecondary antibody washes and incubation in secondary antibody wasperformed in PBS containing 1% bovine serum albumin. A 1:5000 dilutionof PDX1 antibody (gift from Chris Wright, Vanderbilt University) wasused. Flow cytometric analysis was performed substantially as describedin Example 1. The results of the flow analyses are presented in Tables4, 5, and 6

Flow cytometric analyses of either anti-CD55 or CD98 co-stained withanti-PDX1 are shown in Table 4. 15.60% CD55− PDX1+ cells and a total of25.90% PDX1+ cells were present in the population. CD55− cells comprised60.23% of the total PDX1+ cells, making CD55 useful for negativeimmuno-selection of PDX1+ endoderm. Similarly, CD98− cells comprised39.94% of the total PDX1+ cells in the population making CD98 useful fornegative immuno-selection of PDX1+ endoderm.

TABLE 4 CD55 OR CD98 NEGATIVE IMMUNO- SELECTION FOR PDX1 ENDODERM CD55 %of total CD55− CD55+ CD55+ CD55− Total Total PDX1+ cells PDX1+ PDX1+PDX1− PDX1− PDX1+ PDX1− that are CD55− 15.60% 10.30% 47.70% 26.40%25.90% 74.10% 60.23% CD98 % of total CD98− CD98+ CD98+ CD98− Total TotalPDX1+ cells PDX1+ PDX1+ PDX1− PDX1− PDX1+ PDX1− that are CD98− 8.58%12.90% 72.00% 6.52% 21.48% 78.52% 39.94%

Results of flow cytometric analyses of either anti-CD57 or CD142co-stained with anti-PDX1 are shown in Table 5. 21.20% CD57+PDX1+ cellsand a total of 26.10% PDX1+ cells were present in the population. CD57+cells comprised 81.23% of the total PDX1+ cells, making CD57 useful forpositive immuno-selection of PDX1+ endoderm. Similarly, CD142+ cellscomprised 79.30% of the total PDX1+ cells in the population making CD142useful for positive immuno-selection of PDX1+ endoderm.

TABLE 5 CD57 OR CD142 POSITIVE IMMUNO-SELECTION FOR PDX1 ENDODERM CD57 %of total CD57− CD57+ CD57+ CD57− Total Total PDX1+ cells PDX1+ PDX1+PDX1− PDX1− PDX1+ PDX1− that are CD57+ 4.90% 21 .20% 29.10% 44.80%26.10% 73.90% 81.23% CD142 % of total CD142− CD142+ CD142+ CD142− TotalTotal PDX1+ cells PDX1+ PDX1+ PDX1− PDX1− PDX1+ PDX1− that are CD142+4.32% 16.40% 23.60% 55.70% 20.72% 79.30% 79.15%

Results of flow cytometric analyses of either anti-CD55, CD142 and PDX1or CD98, CD142, and anti-PDX1 co-staining are shown in Table 6. TheCD55−CD142+ population was comprised of 83.40% PDX1+ cells and theCD98−CD142+ population was comprised of 90.20% PDX1+ cells. Therefore,either CD55 or CD98 with CD142 can be used to immuno-select for a >80%PDX1+ endoderm cell population.

TABLE 6 COMBINING CD55 OR CD98 NEGATIVE WITH CD142 FOR POSITIVEIMMUNO-SELECTION FOR PDX1 ENDODERM CD55 CD142 CD55− CD55+ CD55+ CD55−CD142+ CD142+ CD142− CD142− 10.40% 22.20% 48.90% 18.50% % of total CD55−CD55+ CD55+ CD55− CD55− CD142+ CD142+ CD142− CD142− Total CD142+ cellsPDX1+ PDX1+ PDX1+ PDX1+ PDX1+ that are PDX1+ 8.67% 10.57% 2.97% 3.20%25.41% 83.40% CD98 CD142 CD98− CD98+ CD98+ CD98− CD142+ CD142+ CD142−CDI42− 8.17% 20.30% 57.90% 13.60% % of total CD98− CD98+ CD98+ CD98−CD98− CD142+ CD142+ CD142− CD142− Total CD142+ cells PDX1+ PDX1+ PDX1+PDX1+ PDX1+ that are PDX1+ 7.37% 7.55% 2.53% 3.67% 21.12% 90.20%

To examine which cell types were marked by the anti-CD142 antibody, hESCdifferentiated stage 5 cultures were analyzed by employingimmunofluorescence cytochemistry using the mouse anti-CD142 antibody (BDPharmingen #550252), a goat anti-PDX1 antibody (gift from Chris Wright),and a rabbit anti-chromogranin A antibody (DakoCytomation). Cellcultures and culture conditions were substantially similar to thosedescribed above in Example 1 and in D'Amour et al. 2005 and 2006 supra.Briefly, cells were washed with PBS and then fixed in 4%paraformaldehyde for 15 minutes at 24 degrees Celsius. Plates werewashed at least 2 times with PBS. Cells were incubated in 5% normaldonkey serum (Jackson Immunoresearch Laboratories, Inc.)/PBS/0.1% TritonX-100 (Sigma) for 15-60 minutes. Cells were then incubated overnight at4 degrees Celsius in 5% normal donkey serum/PBS/0.1% Triton X-100containing the mouse anti-CD142 antibody (BD Pharmingen #550252 at1:20), a goat anti-PDX1 antibody (gift from Chris Wright at 1:2000), anda rabbit anti-chromogranin A antibody (DakoCytomation at 1:200). Cellswere washed at least 3 times in PBS/0.1% Triton X-100, and thenincubated for 1-4 hours at 24 degrees Celsius in 5% normal donkeyserum/PBS/0.1% Triton X-100 containing a 1:500 dilution of the followingfluorochrome conjugated secondary antibodies: donkey anti-mouseAlexaFluor 555 (Invitrogen/Molecular Probes), donkey anti-goatAlexaFluor 488 (Invitrogen/Molecular Probes), and donkey anti-rabbit Cy5(Jackson Immunoresearch Laboratories, Inc.). Cells were washed at least2 times in PBS/0.1% Triton X-100, and then washed at least 2× in PBS.Cell nuclei were identified by incubating the cells for about 10 minutesin PBS containing 1 ug/mL DAPI. Cells were washed 2× in PBS and thenanalyzed using confocal microscopy (Nikon Eclipse 80i, Ci).

Immunofluorescent cytochemistry confirmed the presence of CD142 membranestaining on many PDX1-immunoreactive endoderm cells (green). HoweverCD142 staining was not substantially observed on ChromograninA-immunoreactive endocrine cells.

Example 4 Purification of Endocrine Cells Using CD57, CD142, CD200,CD318, and CD340

To further characterize some of the antibodies described in Example1/Table 1, cells were co-stained with the anti-surface marker antibody(Table 1), an antibody to a known marker of endocrine cells,chromogranin A (CHGA), and/or an antibody to the pancreatic hormonemarker, insulin. CyT49 hES cells were differentiated substantially asdescribed in Example 1. Differentiated cells were then collected on days14 to 22 and stained with anti-surface marker antibody substantially asdescribed in Examples 1 to 3. The following fluorochrome conjugated andunconjugated antibodies were used: CD57−FITC (BD Biosciences #555619),CD142 (BD Biosciences #550252), CD142−PE (BD Biosciences #550312), CD200(BD Biosciences #552023), CD200−PE (BD Biosciences #552475), CD318(Biolegend #324001), CD318−PE (Biolegend #324005), CD340−PE (Biolegend#324406), and CD340−APC (Biolegend #324407). Subsequently, intracellularor intranuclear staining was performed substantially as described inExamples 2 and 3. A 1:100 dilution of anti-CHGA antibody (DAKO #A0430)and a 1:1000 dilution of anti-insulin antibody (DAKO #A0569) were used.Flow cytometric analyses were performed substantially as described inExample 1.

The results of flow cytometric analyses of anti-CD142 or CD340co-staining with anti-CHGA are shown in Table 7. 5.38% CD142−CHGA+ cellsand 6.74% CHGA+ cells were present in the population. CD142− cellscomprised 79.82% of the total CHGA+ cells, making CD142 useful fornegative immuno-selection of endocrine cells. Similarly, CD340− cellscomprised 51.02% of the total CHGA+ cells, making CD340 useful fornegative immuno-selection of endocrine cells.

TABLE 7 CD142 OR CD340 NEGATIVE IMMUNO- SELECTION FOR ENDOCRINE CELLSCD142 % of total CD142− CD142+ CD142+ CD142− Total Total CHGA+ cellsCHGA+ CHGA+ CHGA− CHGA− CHGA+ CHGA− that are CD142− 5.38% 1.36% 35.20%58.00% 6.74% 93.20% 79.82% CD340 % of total CD340− CD340+ CD340+ CD340−Total Total CHGA+ cells CHGA+ CHGA+ CHGA− CHGA− CHGA+ CHGA− that areCD340− 1.75% 1.68% 95.70% 0.82% 3.43% 96.52% 51.02%

Results of flow cytometric analyses of anti-CD57, CD200, or CD318co-stained with anti-CHGA or INS are shown in Table 8. CD57+ cellscomprised 94.01% of the total number of CHGA+ cells, hence CD57 isuseful for positive immuno-selection of endocrine cells. CD200+ cellscomprised 98.26% of total CHGA+ cells making CD200 useful for positiveimmuno-selection of endocrine cells. CD318+ cells comprised 94.49% oftotal CHGA+ cells and 92.04% of total INS+ making CD318 a candidate forpositive immuno-selection of endocrine cells.

TABLE 8 CD57, CD200 OR CD340 POSITIVE IMMUNO- SELECTION FOR ENDOCRINECELLS CD57 % of total CD57− CD57+ CD57+ CD57− Total Total CHGA+ cellsCHGA+ CHGA+ CHGA− CHGA− CHGA+ CHGA− that are CD57+ 0.16% 2.51% 34.50%62.90% 2.67% 97.40% 94.01% CD200 % of total CD200− CD200+ CD200+ CD200−Total Total CHGA+ cells CHGA+ CHGA+ CHGA− CHGA− CHGA+ CHGA− that areCD200+ 0.12% 6.76% 21.50% 71.70% 6.88% 93.20% 98.26% CD318 % of totalCD318− CD318+ CD318+ CD318− Total Total CHGA+ cells CHGA+ CHGA+ CHGA−CHGA− CHGA+ CHGA− that are CD318+ 1.75% 30.00% 20.60% 47.70% 31.75%68.30% 94.49% % of total CD318− CD318+ CD318+ CD318− Total Total INS+cells INS+ INS+ INS− INS− INS+ INS− that are CD318+ 1.28% 14.80% 35.10%48.80% 16.08% 83.90% 92.04%

Example 5 Enrichment of Pancreatic Epithelial/Endoderm Cells andEndocrine Cells Using CD142

To optimize immuno selection of hES-derived pancreatic phenotype cells,CD142 magnetic cell sorting and flow cytometry analysis was performed onvarious hESC differentiated cultures.

Cell cultures and culture conditions were maintained substantially asthat described above in Example 1 and in D'Amour et al. 2005 and 2006supra. hESCs (CyT49) were differentiated to stage 4 to 5 type cells.Cells were briefly washed with PBS. The cells were then enzymaticallydissociated into a single cell suspension using TrypLE at 37 degreesCelsius and then 3% FBS/PBS/1 mM EDTA (sorting buffer) was added. Thesingle cell suspension was passed through a 40-100 uM filter and thenpelleted. Cells were washed in sorting buffer, pelleted and thenresuspended as a single cell suspension in sorting buffer at 1×10(8)cells/mL. Cells were then incubated with Phycoerythrin conjugatedanti-mouse CD142 antibody (BD PHARMIGEN™ Cat. No. 550312) at 10 ul per1×10(7) cells for 15 minutes at 4 degrees Celsius. The cells were washedat least once with 10× volume sorting buffer. Cells were pelleted andresuspended as a single cell suspension at 1×10(8) cells/mL in sortingbuffer containing a 1:5 dilution of anti-PE microbeads (Miltenyi Biotec)and incubated for 15 minutes at 4 degrees Celsius. Cells were washed atleast once with a 10× volume of sorting buffer, pelleted, andresuspended as a single cell suspension at 1×10(8) cells/ml in coldsorting buffer. Immuno-magnetic selection of CD142-positive cells wasthen performed substantially according to manufacturer's instructions(Miltenyi Biotec). Briefly, the single cell suspension was passed over aprewet LS MACS® cell separation column attached to the magneticseparator stand. The column was washed 3 times with 3 mls of sortingbuffer. Cells that did not bind to the column were collected as the flowthrough fraction. The column was removed from the magnetic separatorstand and the bound cells were displaced off the column using 5 mlssorting buffer and cell plunger. The cells in the bound and flow throughfractions were pelleted and fixed in 4% paraformaldehyde for flowcytometry analysis. The pre-sort, bound and flow through fractions werecounter-stained with anti-PDX1 and CHGA substantially as described abovein Examples 3 and 4.

Flow cytometry analysis was performed substantially as described inExample 1. The results of this analysis are shown in Table 9. As shownin Table 9, the bound fraction was highly enriched for CD142+ cellscompared to the pre-sorted and flow through fractions. The boundfraction was also enriched for PDX1+ cells compared to the presort andflow through fractions. The anti-CD142 bound fraction was comprised of71.07% PDX1+ cells compared to 22.26% PDX1+ cells in the pre-sortfraction and 8.32% PDX1+ cells in the flow through fraction. Therefore,there was a 3.19 fold enrichment in PDX1+ cells in the bound fractionrelative to the pre-sort population. In addition the CD142 boundfraction was depleted of CHGA+ endocrine cells compared to the pre-sortpopulation. The pre-sort population was comprised of 11.06% CHGA+ cellswhereas the bound fraction was comprised of 3.67% CHGA+ cells. Most ofthe CHGA+ cells were present in the flow through fraction, which wascomprised of 11.22% CHGA+ cells.

TABLE 9 CD142 IMMUNO-SELECTION OF PANCREATIC EPITHELIAL AND ENDOCRINECELLS CD142+ PDX1+ CHGA+ CD142+ fold PDX1+ fold CHGA+ fold Day 14Pre-sort 38.33% 22.26% 11.06% Bound 82.59% 2.15X 71.07% 3.19X 3.67%0.33X Flow through 24.66% 0.64X 8.32% 0.37X 11.22% 1.01X Day 16 Pre-sort48.40% Bound 83.10% 1.72X Flow through 29.00% 0.60X

To determine which genes were expressed by the CD142 staining cells inthe bound fraction, compared to pre-sorted cells or flow throughfraction cells, total RNA was isolated from the samples with a 6100nucleic acid extractor (Applied Biosystems) and 100-500 ng was used forreverse transcription with iScript cDNA synthesis kit (Bio-Rad). PCRreactions were run in duplicate using 1/40th of the cDNA per reactionand 400 nM forward and reverse primers with QuantiTect SYBR Green mastermix (Qiagen). Alternatively, QuantiTect Primer Assays (Qiagen) were usedaccording to the manufacturer's instructions. Real-time PCR wasperformed using the Rotor Gene 3000 (Corbett Research). Relativequantification was performed in relation to a standard curve. Thestandard curve was created using a mixture of total RNA samples fromvarious fetal human endoderm tissues and differentiated hES cells, and 1μg was used per cDNA reaction in creating the standard curve. Quantifiedvalues for each gene of interest were normalized against the inputdetermined by two housekeeping genes (CYCG and GUSB or TBP). Thestandard deviation of four- or six-gene expression measurements wasreported. Primer sequences: Pdx1 forward primer (5′ to 3′),AAGTCTACCAAAGCTCACGCG (SEQ ID NO: 7); Pdx1 reverse primer (5′ to 3′),GTAGGCGCCGCCTGC (SEQ ID NO:8).

The results of the QPCR analysis are shown in Table 10. Relative foldincreases or decreases in expression of the markers between thedifferent fractions are shown. The expression of pancreatic endodermmarkers including PDX1, NKX6.1, and PTF1A were increased in the CD142bound fraction as compared to the pre-sort and flow through fractions.The expression of endocrine markers including PAX6, INS, and GCG weredecreased in the CD142 bound fraction as compared to the pre-sort andflow through fractions. The expression of pancreatic endoderm markerswere decreased in the CD142 flow through fraction as compared to thepre-sort and bound fractions, whereas the expression of endocrinemarkers was increased in the CD142 flow through fraction as compared tothe pre-sort and bound fractions. CD142 therefore can be used forpositive immuno-selection to enrich pancreatic epithelial/endodermcells, whereas the flow through fraction (the fraction or cells notbinding to the antibody column; or CD142−) is enriched with pancreaticendocrine type cells.

TABLE 10 CD142 ENRICHES FOR PANCREATIC EPITHELIUM/ENDODERM AND/ORPANCREATIC ENDOCRINE TYPE CELLS PDX1 Fold NKX6.1 Fold PTF1A Fold PAX6Fold INS Fold GCG Fold Incease or Increase or Increase or Increase orIncrease or Increase or Decrease Decrease Decrease Decrease DecreaseDecrease Bound/Flow Through 15.32 32.87 21.85 0.18 0.17 0.11Bound/Pre-sort 3.82 8.14 1.95 0.30 0.21 0.17 Flow Through/Pre-sort 0.250.25 0.09 1.69 1.23 1.56

These data demonstrate that CD142 is a useful marker for purifying,enriching and/or isolating for hESC-derived pancreatic epithelial typecells and endocrine cell types.

Example 6 Enrichment of Endocrine Cells Using CD200 and CD318

Similar to that described for CD142 in example 5, characterization ofCD200 and CD318 as novel cell surface markers for hESC-derivedpancreatic endocrine cells from stage 5 cultures was performed byimmuno-selection.

Cell cultures and culture conditions were maintained substantially asthat described herein and in D'Amour et al. 2005 and 2006 supra. hESCs(CyT49) were differentiated to stage 5 type cells and immuno-selectedusing CD200 or CD318 antibody columns. The CD200 and CD318 boundfractions, in some cases, were cultured as cell aggregates for 2 to 4days in either DMEM (Hyclone) or CMRL (Invitrogen) in stage 5 media asdescribed in Example 1. The media was additionally supplemented with 50ug/mL DNase, 2% FBS, and 10 ng/mL FGF2. Cell aggregates were dissociatedto a single cell suspension using TrypLE and processed for flowcytometry analysis substantially as described in Example 5.

Flow cytometry was performed substantially as described above in Example5. In this case, intracellular cellular staining was performed withanti-CHGA and anti-INS antibodies on the pre-sort, bound, boundaggregate, and flow through fractions as described in Examples 2-5.Results of this analysis are shown in Table 11. Immuno-selection withCD200 enriched for CHGA+ and INS+ endocrine cells as shown by a 1.89×increase in CHGA+ cells and a 2.43× increase in INS+ cells in the CD200bound fraction (67.5% CHGA+ cells, 19.9% INS+ cells) as compared to thepre-sort fraction (35.7% CHGA+ cells, 8.2% INS+ cells). Furtherenrichment of CHGA+ and INS+ cells was observed upon aggregation of theCD200 bound cells. The CD200 bound aggregates were about 84% CHGA+(2.35×enrichment) and 22.5% INS+(2.74× enrichment). Similar enrichment inCHGA+ endocrine cells (2.91× enrichment in the CD200 bound fraction) wasobserved in a second CD200 immuno-selection experiment. Hence,immuno-selection with CD200 enriched for CHGA+ endocrine type cells. TheCD318 bound fraction was 2.08× enriched in CHGA+ type cells (71.0% CHGA+cells) as compared to the pre-sort fraction (34.1% CHGA+). Again,further enrichment of CHGA+ cells was observed upon aggregation of theCD318 bound fraction. The CD318 bound aggregates were 93.9% CHGA+(2.75×enrichment).

TABLE 11 ENRICHMENT OF ENDOCRINE CELLS USING IMMUNO-SELECTION WITH CD200AND CD318 Bound Aggregate Pre-sort Bound Bound Endocrine Bound AggregateEndocrine Fold Endocrine % Endocrine % Fold Enrichment Endocrine %Enrichment CD200 CD200+ CD200+ CHGA = CHGA+ = CHGA = CHGA+ = CHGA+ =1.89X 84.0% 2.35X 35.7% 67.5% CD200+ CD200+ INS = INS+ = INS = INS+ =INS+ = 2.43X 22.5% 2.74X  8.2% 19.9% CD200 CD200+ CD200+ CHGA = n.d.n.d. CHGA+ = CHGA+ = 2.91X 11.4% 33.2% CD318 CD318+ CD318+ CHGA = CHGA+= CHGA = CHGA+ = CHGA+ = 2.08X 93.9% 2.75X 34.1% 71.0%

Expression of CD200 on endocrine cells was further characterized byperforming immunofluoroscence cytochemistry substantially as describedin Example 3 for CD142. Cell surface staining of CD200 on CHGA+ cellswas observed in stage 5 differentiated cultures.

In view of the foregoing data, there is a sub-population of hES-derivedpancreatic endoderm type cells, in particular, pancreatic epithelium andpancreatic endocrine phenotypes, which are positive for CD142 and CD200,respectively. The CD142 staining pancreatic epithelial cells wereco-positive or co-stained with some Pdx1 but not with CHGA+ stainedcells, which cells are characteristic of pancreatic endocrine cells atthis stage of differentiation. Conversely, CD200 staining pancreaticendocrine cells were co-positive or co-stained with mostly CHGA+ cellsbut not with PDX1 epithelium stained cells. These data indicate that atleast CD142 and CD200 are markers which to date have not been describedto enrich, isolate, deplete, separate, sort and/or purify humanpancreatic epithelium and pancreatic endocrine phenotype cells. Suchmarker(s) provide a method for purifying hES-derived cell types fortherapeutic applications.

Example 7 Pancreatic Endoderm Cell Cultures

Human ES cells were differentiated to late stage 4 early stage 5 andthen analyzed by flow cytometry using anti-PDX1−APC (R&D Systems#IC2419A), anti-CHGA, and anti-NKX6.1 antibodies as described above.Using this combination of markers the composition of the following celltypes in hES-derived cultures can be identified: Pancreatic endoderm(CHGA−PDX1+NKX6.1+), endocrine cells (CHGA+), and PDX1+ endoderm(CHGA−PDX1+NKX6.1−). Because PDX1+ and NKX6.1+ cells are also expressedin some endocrine cells it is necessary to use CHGA to furtherdistinguish between these pancreatic cell lineages (i.e., pancreaticendoderm vs. pancreatic endocrine cells). However, during embryonicdevelopment, only the pancreatic endoderm is expected to containCHGA−PDX1+NKX6.1+ cells.

As shown in Table 12 and FIG. 2 most of the hES-derived cells in stage 4to 5 cultures can be identified using the indicated 3 markers. Theculture was comprised of 14.08% CHGA+ endocrine cells. As expected somethese CHGA+ cells were also positive for PDX1+ and NKX6.1. The culturewas also comprised of 57.61% CHGA−PDX1+NKX6.1+ pancreatic endoderm cellsand 21.00% CHGA−PDX1+NKX6.1− cells. Thus, cell cultures enriched inpancreatic endoderm can be obtained. The pancreatic endoderm cells canbe further purified using any of the above immuno selection methodsdescribed above, e.g., CD142 immuno selection further purifiespancreatic endoderm from other cell populations such as endocrine cells.

TABLE 12 PANCREATIC ENDODERM AND ENDOCRINE CELL TYPES IN STAGE 4-5HES-DERIVED CULTURES Pancreatic Endoderm CHGA− CHGA− CHGA− CHGA− TotalNKX6.1+ NKX6.1− NKX6.1+ NKX6.1− CHGA− PDX1+ PDX1+ PDX1− PDX1− 85.8057.61 21.00 4.08 3.04 CHGA+ CHGA+ CHGA+ CHGA+ Total NKX6.1+ NKX6.1−NKX6.1+ NKX6.1− CHGA+ PDX1+ PDX1+ PDX1− PDX1− 14.08  3.35 8.90 0.34 1.49Total Total PDX1+ NKX6.1+ 90.86 65.38

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications can be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

For example, the surface markers discussed herein can be used eitheralone or in combination with each other, or any of the cell surfacemarkers as described in Table 1 (Example 1). Therefore, anti-CD30,CD49a, CD49e, CD55, CD99, CD165, and CD334 can be used to enrich foregutendoderm. CD55, CD57, CD98, and CD142 can be used to enrich pancreaticendoderm. CD57, CD142, CD200, CD318, and CD340 can be used to enrichendocrine cells. These and other embodiments are encompassed by thepresent invention.

Also, at least FOXA2 is expressed in definitive endoderm as well as inStage 2 gut endoderm described herein (Table 2, Example 2), hence thesame antibodies which were used to demonstrate immuno-selection(positive and/or negative) can be employed to enrich or purify fordefinitive endoderm. This same principle can be applied to posteriorforegut endoderm (Stage 3), which also expresses PDX1 similar topancreatic endoderm (Stage 4). Therefore, the antibodies which were usedto demonstrate enrichment and purification for pancreatic endoderm canbe employed to enrich and purify for posterior foregut.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

What is claimed is:
 1. A method of enriching for pancreatic lineagecells comprising: a) exposing an in vitro cell population comprisingpancreatic lineage cells to a ligand that binds a cell-surface marker,wherein the cell-surface marker is CD200, CD318, CD142, CD57 or CD340;and b) selecting cells that bind to said ligand that binds thecell-surface marker, wherein the cell-surface marker is CD200, CD318 orCD57 or selecting cells that do not bind to said ligand that binds themarker, wherein the marker is CD142 or CD340, thereby enriching forpancreatic lineage cells.
 2. The method of claim 1, wherein the enrichedpancreatic lineage cells are insulin-positive cells.
 3. The method ofclaim 1, wherein the enriched pancreatic lineage cells are pancreaticendocrine cells.
 4. The method of claim 1, wherein said ligand comprisesan antibody or binding fragment thereof that specifically binds thecell-surface marker.
 5. The method of claim 1, wherein said ligand isassociated with a detectable label.
 6. The method of claim 1, whereinsaid ligand is associated with a magnetic particle.
 7. The method ofclaim 1, wherein the pancreatic lineage cells are selected byFluorescence Activated Cell Sorting (FACS).
 8. A method of enriching forpancreatic endocrine cells comprising: a) exposing an in vitro cellpopulation comprising insulin positive cells to a ligand that binds acell-surface marker, wherein the marker is CD200 or CD318; and b)selecting cells that bind to said ligand that binds the cell-surfacemarker, thereby enriching for pancreatic endocrine cells.
 9. The methodof claim 8, wherein the enriched pancreatic endocrine cells expressinsulin.
 10. The method of claim 8, wherein said ligand comprises anantibody or binding fragment thereof that specifically binds thecell-surface marker.
 11. The method of claim 8, wherein said ligand isassociated with a detectable label.
 12. The method of claim 8, whereinsaid ligand is associated with a magnetic particle.
 13. The method ofclaim 8, wherein the pancreatic endocrine cells are selected byFluorescence Activated Cell Sorting (FACS).
 14. A method of enrichingfor cells expressing insulin comprising: a) exposing an in vitro cellpopulation comprising cell expressing insulin to a ligand that binds acell-surface marker, wherein the cell surface marker is CD200 or CD318;and b) selecting cells that bind to said ligand that binds thecell-surface marker, thereby enriching for cells expressing insulin. 15.The method of claim 14, wherein said enriched cells expressing insulinare pancreatic endocrine cells.
 16. The method of claim 14, wherein saidligand comprises an antibody or binding fragment thereof thatspecifically binds the cell-surface maker.
 17. The method of claim 14,wherein said ligand is associated with a detectable label.
 18. Themethod of claim 14, wherein said ligand is associated with a magneticparticle.
 19. The method of claim 14, wherein the cells expressinginsulin are selected by Fluorescence Activated Cell Sorting (FACS). 20.The method of claim 14, wherein the cells expressing insulin are cellaggregates.